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Volume 81 (1977)
- Issue 6 Pages 1583-
- Issue 5 Pages 1181-
- Issue 4 Pages 813-
- Issue 3 Pages 533-
- Issue 2 Pages 277-
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Volume 81, Issue 4
Displaying 1-46 of 46 articles from this issue
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Masayoshi ASATANI, Kiyoshi KURAHASHI1977 Volume 81 Issue 4 Pages 813-822
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus brevis ATCC 9999, which produces gramicidin S, was found to be unable to ferment various carbohydrates such as glucose, galactose, mannose, lactose, maltose, sucrose, and mannitol. This defect in carbohydrate utilization was attributed to the lack of the transport system (s) for various carbohydrates. This organism can take up only fructose and glycerol and form acids. The presence in this organism of the phosphoenolpyruvate: sugar phosphotransferase system for fructose, but not glycerol, as well as kinases for fructose and glycerol was demonstrated.View full abstractDownload PDF (916K) -
Shin-ichi SUGIMOTO, Isamu SHIIO1977 Volume 81 Issue 4 Pages 823-833
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen tryptophan auxotrophs derived from Brevibacterium flavuin which lacked tryptophan synthetase-A (TS-A), tryptophan synthetase-B (TS-B) or anthranilate-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT), were cultured in media containing limiting or excess tryptophan, the specific activities of all six tryptophan enzymes were much higher under conditions of limiting tryptophan than under conditions of excess tryptophan. This indicates that the formation of all the tryptophan enzymes was repressed by tryptophan. However, the ratio of specific activities under the two conditions was not constant among these enzymes. When the rates of synthesis of the enzymes during tryptophan starvation were compared, those of anthranilate synthetase (AS), N-5'-phosphoribosylanthranilate isomerase (PRAI), TS-A and TS-B were coordinate, while the specific activity of indole-3-glycerol phosphate synthetase (InGPS) did not increase significantly. In order to examine the molecular states of tryptophan enzymes, enzyme extracts of tryptophan auxotrophs were subjected to get filtration on a Sephadex G-200 column. The elution profiles gave four distinct peaks; AS, PRT, PRAI and InGPS, TS-A and TS-B. Although PRAI and InGPS activities were completely coeluted in the gel filtration, they were partially dissociated on DEAE-cellulose column chromatography, giving three peaks of activity for PRAI, InGPS, and PRAI plus InGPS. TS-A and TS-B were only slightly separated in Sephadex G-200 gel filtration with glycerol. On the other hand, in the gel filtration without glycerol, neither TS-A nor TS-B activity was detected in the eluate. However, when TS-A activity was assayed in the presence of TS-B, TS-A was detected but showed a much lower molecular weight than that observed in the gel filtration with glycerol. This suggested that TS-A and TS-B were dissociated in the absence of glycerol. That is, among the six tryptophan enzymes, two dissociable enzyme complexes were observed; one included TS-A and TS-B, the other PRAI and InGPS.View full abstractDownload PDF (846K) -
I. Purification and Identification of Copper Coproporphyrin IIITakako YAMADA-ANKEI, Hidekazu IWASAKI, Takeshi MORI1977 Volume 81 Issue 4 Pages 835-842
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus cereus strains 2 and T did not form spores and accumulated a large amount of purple pigment inside the cells, when cultured in a yeast extract-ammonium salt medium with excess glucose. The pigment was extracted and crystallized as the ethyl ester. It was identified as copper coproporphyrin III.View full abstractDownload PDF (951K) -
II. Regulation of the Biosynthesis of Coproporphyrin III and Its Copper Complex by Oxygen and Heavy Metal IonsTakako YAMADA-ANKEI, Hidekazu IWASAKI, Takeshi MORI1977 Volume 81 Issue 4 Pages 843-850
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of oxygen and heavy metal ions on the production of copper coproporphyrin III were studied in Bacillus cereus strain 2.
The formation of copper coproporphyrin III was found to be maximum when the cells were cultivated in G-medium at a low level of oxygen supply, but it was suppressed at extremely low oxygen supply levels.
When the cells were cultured in metal-free G-medium, neither metal-coproporphyrin III nor coproporphyrin III was formed. In the presence of copper in the medium (400-100 μM), the formation of coproporphyrin III copper salt was maximum, but the addition of various heavy metal ions other than copper to the copper-free medium resulted in the formation of neither coproporphyrin III nor its metal chelates. Copper ions appear to be specifically required for coproporphyrin III formation.View full abstractDownload PDF (495K) -
Toshie TOKIEDA, Toshio NIIMURA, Fuminori TAKAMURA, Tsutomu YAMAHA1977 Volume 81 Issue 4 Pages 851-858
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80, 000 by gel filtration. The enzyme catalyzed the following reaction:
cyclohexylamine+O2+H2O→eyclohexanone+NH3+H2O2
The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5×10-4 M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases.
The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observations that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.View full abstractDownload PDF (623K) -
Hirofumi OGAWA, Hiroshi SHIRAKI, Yoshihiro MATSUDA, Kinji KAKIUCHI, Ha ...1977 Volume 81 Issue 4 Pages 859-869
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAdenylosuccinate synthetase [EC 6.3.4.4] was purified and crystallized from rat skeletal muscle. The purified preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and ultracentrifugal analysis, and by the latter procedure its average molecular weight was estimated to be 104, 000 and its S20, W value, 5.0.
On SDS-polyacrylamide gel electrophoresis it gave only a single band with a molecular weight of 52, 000. Sedimentation equilibrium experiments indicated that its molecular weight in 6M guanidine-HCl was 49, 000. Amino acid analyses indicated a subunit molecular weight of 52, 860. These findings show that the native enzyme is composed of two subunits with molecular weights of 52, 000.
The optimum pH was 6.8 and the Km values for L-aspartate, IMP, and GTP were calculated to be 2.5, 7.0, and 3.8×10-4M, respectively.
Nucleoside mono- and diphosphates, whether purine or pyrimidine derivatives, were potent inhibitors, while nucleoside triphosphates were not.
The enzyme activity was inhibited considerably by succinate derivatives such as succinate, adenylosuccinate, and argininosuccinate. Succinate caused competitive inhibition with respect to aspartate with a K1 value of 4×10-4M, and noncompetitive inhibition with respect to IMP and GTP with Ki values of 7.7×10-3 M and 2.6×10-3M, respectively.
The purified enzyme preparation showed full activity in the presence of MgCl2, and MgCl2 could be partially replaced by MnCl2 CoCl2, or CaCl2.View full abstractDownload PDF (1399K) -
Effects of Detergents on the Conformation of Escherichia coli tRNA as Measured by Circular DichroismNobuo OKABE, Michiyo SERIKAWA, Ken-ichi TOMITA1977 Volume 81 Issue 4 Pages 871-877
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of a cationic detergent, lauryl pyridiniumchloride (LPC), and an anionic one, sodium n-octylbenzenesulfonate (SOBS), on the conformation of unfractionated Escherichia coli tRNA was investigated at various molar ratios of detergent to tRNA (D/R) in the presence and absence of Mg2+ and Na+ ions by measuring the circular dichroism (CD) at 265nm and 340 nm, which reflects conformational change involving base pairs and/or base stacking, and the disymmetry in the vicinity of 4-thiouridylate (4-TU), respectively.
In the presence of Mg2+ and Na+ ions, the tRNA retains its native structure even in the presence of high molar ratios of detergent to tRNA (D/R_??_40 at 265nm and D/R_??_20 at 340 run). However, in the absence of these metal ions, the ellipticity at 340nm was very sensitive to LPC concentration and decreased from 5, 600 to nearly -1, 600 at 25°C with the increase of D/R ratios up to 20, and a similar decrease in the ellipticity at 340nm was observed on thermal denaturation. This result suggests that the local environment involving the 4-TU region might be readily influenced by LPC, reflecting a large conformational change. However, no effect was observed in the case of the SOBS: tRNA system. On the other hand, secondary base pairing and/or base stacking structure was virtually invariant on adding both LPC and SOBS even at high D/R ratios in the absence of Mg2+ and Na+ ions.View full abstractDownload PDF (465K) -
Shogo ICHII, Noriko MURAKAMI, Masao IZAWA1977 Volume 81 Issue 4 Pages 879-884
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytoplasmic 3H-5α-dihydrotestosterone-receptor complex of the rat ventral prostate was efficiently taken up by nuclei isolated from the liver, the ventral prostate and the spleen. The amount of the complex bound to liver nuclei was approximately 3 times that to prostate nuclei, and a relatively small amount of the complex was taken up by spleen nuclei under the incubation conditions: 0.16nM complex (2.0mg as protein) was used. Binding of the complex to liver nuclei was low-affinity and non-saturable, while in nuclei from the ventral prostate and the spleen, high-affinity and saturable binding was observed. Kd of nuclear binding in the latter two tissues was roughly of the order of 10-70M. Castration caused a reduction in the binding of the complex to prostate nuclei and replacement therapy with testosterone propionate completely blocked the effect of castration on the nuclear binding. The affinity of high-affinity binding was not changed significantly after castration. No significant effects of castration and testosterone treatment were observed in the binding of nuclei from the other two tissues examined.
The physiological significance of the nuclear binding of hormone-receptor complexes in the action of steroid hormones is discussed.View full abstractDownload PDF (453K) -
Noriko MURAKAMI, Shogo ICHII1977 Volume 81 Issue 4 Pages 885-889
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo gain insight into the mechanism of age-related changes in tissue responsiveness to glucocorticoids, the binding patterns of dexamethasone (Dex) to cytoplasmic receptor and of cytoplasmic Dex-receptor complex to nuclei were compared in the livers of adult, 7-day-old, newborn and fetal rats. Only one binding component with high binding affinity was observed in the cytosol from the fetal liver, while at least 2 binders which showed high and low affinity to Dex were detected in the cytosol of the adult liver. Kd of the high-affinity binder in the cytosol of fetal liver was different from that in the adult liver; 3.6×10-9M for fetal and 9.1×10-10M for adult. Cytoplasmic Dex-receptor complexes from the adult and fetal liver were efficiently bound to nuclei from adult, 7-day-old, newborn and fetal livers. The amount of complex bound to nuclei of the adult liver was largest and the least binding was observed in nuclei of the newborn liver under the incubation conditions used, with 1.77nM 3H-Dex-receptor complex. A high-affinity binding of the receptor complex from the adult liver was observed in fetal nuclei (Kd =1×10-9M), but nuclei from the other tissues showed low-affinity and non-saturable binding. The Dex-receptor complex from the fetal liver bound to nuclei of the adult liver in a low-affinity and non-saturable manner, while high-affinity binding (Kd=6×10-10M) was observed in nuclei of the fetal liver. The binding of the fetal complex to nuclei of the 7-day-old and newborn livers showed a transitional binding pattern, with sites of high- and low-affinity binding coexisting. Based on these results, it was concluded that differences in the hepatic cytosol receptor-nuclear acceptor system may be attributable, at least in part, to age-related changes in tissue responsiveness to glucocorticoids.View full abstractDownload PDF (340K) -
II. Further Studies on Two Groups of Anti-MANS Antibodies with Significantly Different Effects on MANSamide Fluorescence Produced by a Single RabbitMasahiko ONODERA, Hiroyuki SHIOKAWA1977 Volume 81 Issue 4 Pages 891-898
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs described previously, a single rabbit (No. 13) produced two groups of anti-MANS antibodies with significantly different effects on MANSamide fluorescence. These groups, anti-MANS antibody (415nm) and anti-MANS antibody (465nm), had emission maxima at 415nm and 465nm, respectively.
This paper deals with the immunochemical properties of the two groups of antibodies
(1) The results of fluorometric titration of these groups with MANSamide indicated that anti-MANS antibody (465nm) had a higher affinity for MANSamide than did anti-MANS antibody (415nm).
(2) More than 70% of anti-MANS antibody 415nm) was adsorbed on Sepharose covalently coupled with bovine serum albumin, which was used as a carrier during immunization, while a half of anti-MANS antibody (465nm) was adsorbed.
(3) Only one of the groups, anti-MANS antibody (465nm), had precipitating activity toward MANS-rabbit serum albumin used as a test antigen.View full abstractDownload PDF (1459K) -
Sanae MII1977 Volume 81 Issue 4 Pages 899-909
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lag phase in the polymerization of ribonucleoside 5'-diphosphate by polynucleotide phosphorylase from Azotobacter vinelandii was found to be caused by a basic protein isolated from the crude enzyme preparation or by a synthetic basic polypeptide. Poly(A) formed during the lag phase was bound to added polylysine in a 1:1 ratio on a monomer basis. Preferential repression of polymerization of UDP was observed with polylysine and of ADP with polyarginine. These findings may explain why the polymerization of UDP is slower than that of ADP, and suggest that a lysine-rich area in the enzyme may act as a regulatory site. The so-called primer can thus be considered as an activator.
The lag phase in UDP polymerization caused by polylysine was eliminated by poly(U) or poly(C) and prolonged by poly(A), in a manner similar to that previously observed with the priming specificity. A lag phase was also caused by polylysine in the P1-ADP exchange reaction, but not in poly(A) phosphorolysis. The effect of basic polypeptide on the nucleotidyltransferase activity of the enzyme indicates that the complete reaction of Azotobacter polynucleotide phosphorylase is an exchange reaction, which includes polymerization and phosphorolysis. A complex, [polynucleotide phosphorylase-Mg2+-ribonucleoside 5'-diphosphate], which the enzyme appears to form as a reactive intermediate, and a possible scheme for the priming specificity are described.View full abstractDownload PDF (847K) -
Takeshi TSUJIMOTO1977 Volume 81 Issue 4 Pages 911-921
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reduction of NAD(P) by ADP, a reversal of the normal change, in rat kidney mitochondria incubated in isotonic media containing NaCI and EDTA was studied by dual-wavelength spectrophotometry. Dual-wavelength spectrophotometry and respiratory studies showed that with increasing concentrations of NaCl and EDTA, rapid respiratory rate and marked oxidation of NAD(P) became evident in state 4, respiratory stimulation by ADP was reduced considerably, and the oxidation-reduction pattern of NAD(P) by ADP was converted to a diminished initial oxidation and subsequent reductive-reoxidative changes. Spectroscopic and enzymatic assays confirmed that the NAD(P) reductive change by ADP observed in dualwavelength spectrophotometry appeared to coincide with a remarkable swelling-shrinkage reaction larger in magnitude than the diminished oxidation of NAD by ADP. Under experimental conditions which might involve a significant swelling-shrinkage reaction, the redox kinetics observed by dual-wavelength spectrophotometry do no show the true NAD+: NADH change. The marked swelling-shrinkage was ascribed to an increase of ionic permeability due to the chelation by EDTA of membrane-bound magnesium with loss of potassium and of the integrity of mitochondria. ADP-induced inhibition of respiration and reduction of NAD, which will be designated here as “true reverse acceptor control, ” was not observed. Instead, the rapid respiratory rate in state 4, the decline of respiratory control, oxidized NAD in state 4, inhibition of further oxidation of NAD in state 3, and the small phosphorylation deficiency observed in the NaCl-EDTA media, suggested that a leaky or loosely coupling state rather than uncoupling led to the apparent reduction.View full abstractDownload PDF (877K) -
Norio OOKUBO1977 Volume 81 Issue 4 Pages 923-931
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe oxidation of rabbit skeletal tropomyosin (TM) by repeated cycles of freezing and melting in 0.3mM Na bicarbonate was studied by electrophoresis and column chromatography.
The oxidized TM showed two bands at ca. 70, 000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Each band component was separated into disulfide-linked all and as by carboxymethyl cellulose (CMC) column chromatography in urea. Oxidized TM before fractionation, as well as the αβ and αα components, was found to have a molecular weight of about 80, 000 daltons, indicating the disulfide bonds to be primarily intramolecular. Oxidation of dilute TM in 1M NaCl by exposure to air also produced disulfide-linked αβ.
Partially oxidized TM was found to separate into β, αβ, α, and αα on CMC chromatography, and these were eluted with a linear gradient of NaCl at molarities of ca. 0.09, 0.11, 0.12, and 0.14M, respectively. The oxidation process was investigated by CMC chromatography, , and a possible mechanism is presented. The αβ and as components may exist as dominant components in TM in vitro rather than as a random mixture of two subunits.
A splitting of the electrophoretic band of the α subunit into a doublet was observed.View full abstractDownload PDF (2982K) -
II. Effect of Modification of the Sa Thiol Group on Superprecipitation and ClearingTatsuhisa YAMASHITA, Tsuneyoshi HORIGOME1977 Volume 81 Issue 4 Pages 933-939
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of Sa modification with NEM, which activates Mg2+-ATPase through an enhancement of the association of actin and myosin, was investigated on the superprecipitation, clearing and Mg2+-ITPase of myosin B with reference to the effect of S1-blocking.
1. Superprecipitation induced by ATP was markedly enhanced by Sa-blocking even at high concentrations of Mg2+ and substrate; this may be due to an increase in the affinity of myosin and actin on blocking Sa.
2. Nevertheless, neither ITP-induced superprecipitation nor Mg2+-ITPase was affected by Sa modification.
3. Blocking of S1 brought about the inhibition of ATP- and ITP-induced superprecipitation and Mg2+-ITPase activity, suggesting that Sa-blocking decreases the affinity of myosin and actin.
4. Sa-blocked myosin B showed greater resistance to clearing by ATP, especially in the presence of Ca2+ ions, whereas in the clearing response of actomyosin gel to PP1 no difference between Sa-blocked and unmodified myosins B was observed. On the other hand, the clearing response of myosin B became more sensitive to both ATP and PP1 on blocking S1.
Based on the above results and preliminary data suggesting that Sa is located in LMM, the interaction of myosin filaments and actin filaments under physiological conditions is discussed.View full abstractDownload PDF (522K) -
Masao ONO, Toshihiko KONDO, Masaya KAWAKAMI1977 Volume 81 Issue 4 Pages 941-947
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolysomes producing IgGl(k) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410, 000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25, 000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cellfree system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Cτt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4.4-fold during the immunoprecipitation of polysomes.View full abstractDownload PDF (1916K) -
Masao ONO, Toshihiko KONDO, Masaya KAWAKAMI, Tasuku HONJO1977 Volume 81 Issue 4 Pages 949-954
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700, 000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55, 000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogeneous with rabbit globin mRNA as a standard.View full abstractDownload PDF (1532K) -
Atsushi IKAI, Hitoshi YAGISAWA1977 Volume 81 Issue 4 Pages 955-961
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteolytic treatment of human serum low density lipoprotein (LDL) resulted in the observation of interesting time-dependent changes in the sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of apo-LDL. Five major fragments with well-defined relative mobilities appeared within 30 min of protease treatment. Prolonged treatment with subtilisin caused changes in the amount of peptides in each of the five bands but their positions on the gel remained unchanged. Periodic acid-Schiff base staining of the gel showed a proteolytic fragment with an apparent molecular weight of 110, 000 (actually a cross-linked dimer of two peptides with molecular weights of 77, 000 and 68, 000) to be a carbohydrate-bearing peptide that was most resistant to further proteolysis and therefore responsible for the interaction between the digested LDL and concanavalin A.View full abstractDownload PDF (2472K) -
Yoshiyuki TANAKA, Ikuzo URITANI1977 Volume 81 Issue 4 Pages 963-970
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Phenylalanine ammonia-lyase (PAL) activity was developed in response to cut injury in sweet potato root tissue. The enzyme was purified from tissue incubated for I day after slicing by ammonium sulfate fractionation, column chromatographies on L-phenylalanyl Sepharose 4B, phosphocellulose, Sephadex G-200 and Sepharose 6B and preparative polyacrylamide gel electrophoresis. The molecular weight and sedimentation coefficient were estimated to be 285, 000 to 320, 000 and 11.6 to 11.9 S, respectively. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel yielded a single stained protein band which corresponded to a subunit weight of 80, 000. Thus, the enzyme seems to be composed of four subunits of the same size. Neither L-tyrosine nor D-phenylalanine served as a substrate. Two Km values for the PAL were observed above and below 30μM at various temperatures and were lower than those for PALs of other plants. The slope of the Arrhenius plot had a discontinuity at 17°C. The values of activation energy were calculated to be 15, 000 cal and 19, 000 cal above and below 17°C, respectively. Similar discontinuities were also observed in the effect of temperature on the Km values and the Hill coefficients. Negative cooperativity was observed at 10°C (n=0.83), but was not marked above 20°C (n=0.94).View full abstractDownload PDF (1020K) -
Shintaro TAKEUCHI, Akio MAEDA1977 Volume 81 Issue 4 Pages 971-976
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of fluorescein mercuric acetate (FMA) with thiol groups is inhibited by barbital (Veronal). However, barbital has no effect on the reaction of 5, 5'-dithiobis(2-nitrobenzoic acid) with thiol groups. Complex formation between FMA and barbital was shown by an increase in the fluorescence polarization of FMA in the presence of barbital. These findings indicate that the inhibitory effect of barbital is due to its interaction with FMA. The extent of this inhibition, which depends on the nature of the thiol-containing substance, presumably reflects the structure surrounding the thiol groups. Furthermore, the absorption and fluorescence properties of FMA conjugated with thiol groups are sensitive to the environment. Because of these properties, FMA can be used to investigate the environment of thiol groups.View full abstractDownload PDF (473K) -
Koichi YAGI, Hideto KUWAYAMA1977 Volume 81 Issue 4 Pages 977-988
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubfragment-1 was prepared from pig cardiac myosin by chymotryptic digestion at low ionic strength at 0-2°C for 20 h. Two components were distinguished in polyacrylamide gel electrophoresis of subfragment-I preparations, and were designated as S-1 (CT) and S-1 (CT)'. The isoelectric points were 6.70 and 6.45, respectively. The two components could not be separated by Sephadex G-200 gel filtration, but they were separated on a DEAE-cellulose (DE 32) column or a 6-aminohexylPP1-Sepharose 4B conjugate column. S-1 (CT) consisted of one large polypeptide chain (the head portion of the f-chain) and one small polypeptide chain (g1). In SDS gel electrophoresis, the large polypeptide of S-1 (CT) migrated at the same rate as that of S-1 (CT)', but the small polypeptide of S-1 (CT)' migrated at a faster rate than that of S-1 (CT). The small chain of S-1 (CT)' was thus designated as g1'. The migration rate of the small chain of S-1 (CT) was the same as that of g1 of cardiac myosin, but the small chain of S-1 (CT)' migrated faster than g1 and slower than g2 of cardiac myosin. It was therefore suggested that S-1 (CT)' was produced by overdigestion of S-1 (CT).
The yield of S-1 (CT) plus S-1 (CT)' estimated on the basis of absorption at 280nm was 36% of the weight of myosin employed. The molecular weight was 1.19×105 and the maximum ADP binding number was 0.48 mol using S-1 (CT) 30% contaminated by S-1 (CT)'. The pH-activity relationships in the presence of Ca2+ and EDTA were investigated. The ATPase activity in the presence of Ca2+ at pH 7.5 was 0.3 pmol mg-1-min-1 for both S-1 (CT) and S-1 (CT)'. The maximum ATPase activity of S-1 (CT) in the presence of F-actin was 8.3 jtmol mg-1. min-1 and the affinity for F-actin was about 1/20 of that of skeletal S-1 (CT).View full abstractDownload PDF (1947K) -
Kinzo NAGASAWA, Tsugumi TOKUYASU, Yuko INOUE1977 Volume 81 Issue 4 Pages 989-993
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive partially N-desulfated heparin preparations (N-sulfate: 0.73-0.48 mol, O-sulfate: 1.33-1.29 mol, anticoagulant activity: 134-102 USP units) were prepared by solvolytic N-desulfation of the pyridinium salt of hog mucosal heparin (N-sulfate: 0.83 mol, O-sulfate: 1.42 mol, N-acetyl: 0.12 mol, anticoagulant activity: 165 USP units) in DMSO containing 5% water. The preparations, ranging in degree of N-desulfation from 12 to 31%, which were obtained by reaction at 20°C for 5-60 min, retained an average of 79% of the original activity. A preparation with a degree of N-desulfation of 42%, which was obtained by reaction at 20°C for 120 min, retained 62% of the original activity.
Five N-acetylated heparin preparations (N-acetyl: 0.17-0.49 mol) were prepared by N-acetylation of the partially N-desulfated heparins. Their anticoagulant activities (133-107 USP units) were nearly equal to those of the corresponding starting N-desulfated heparins.
N-Succinoylation of a partially N-desulfated heparin (N-sulfate: 0.57 mol, anticoagulant activity: 125 USP units) did not enhance its biological activity, but N-resulfation restored its anticoagulant activity to 94% of that of the original heparin.
Partially N-acetylated heparin with 0.24 mol of N-acetyl groups, two whale heparin preparations, and a purified heparan sulfate fraction, all of which had similar N-acetyl contents (0.24-0.27 mol), were assayed for anticoagulant activity, and the correlation between biological activities and chemical properties is discussed.View full abstractDownload PDF (353K) -
III. Detection of Single-Stranded Regions by Digestion with Nuclease S1Kazuya NISHIKAWA, Shosuke TAKEMURA1977 Volume 81 Issue 4 Pages 995-1003
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIdentification of single-stranded regions in Torulopsis utilis 5S RNA was attempted by the use of Nuclease S1, a single-strand specific endonuclease. When T. utilis 5S RNA was subjected to prolonged incubation with Nuclease S1, about 50% of the substrate 5S RNA remained as large oligonucleotide “cores.” Such Nuclease S1-resistant fragments were purified and sequenced by column chromatographic procedures. These analyses revealed that regions around positions 12, 40, 57, and 110 are in exposed single-stranded loops at 37°C and that regions around positions 12 and 40 are most exposed at 20°C. These results are compatible with our secondary structure model for T. utilis 5S RNA (Nishikawa & Takemura (1974) J. Biocheni. 76, 935-947) except that the 5' part of the molecule (from the region around position 22 to that around position 57) might have a somewhat looser conformation than our secondary structure model suggests. The implications of such results are also discussed in relation to the presumed function of the sequence C-G-A-U-C (around position 40) as one of the recognition sites for initiator tRNA binding on ribosomes.View full abstractDownload PDF (634K) -
Yukio YASUKOCHI, Michio NAKAMURA, Shigeki MINAKAMI1977 Volume 81 Issue 4 Pages 1005-1009
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The amount of cytochrome b5 was not changed significantly by a single injection of cobalt (60mg/kg body weight) or by daily injection of cobalt (30mg/kg body weight) for 4 days or 8 days. On the other hand, the amount of cytochrome P-450 was depressed strongly by both cobalt treatments.
2. The incorporations of [3H] leucine as well as 5-amino [3H] Ievulinate into cytochrome b5 in cobalt-treated animals were almost the same as those in the controls 5 h after injections of the radioisotopes, whereas the radioactivity of heme labelled with 5-amino [3H] levulinate in microsomal residues after trypsin digestion, which would consist mainly of cytochrome P-450, was higher in the controls than in cobalt-treated animals after 5 h.View full abstractDownload PDF (381K) -
Mitsuo NAKAGAWA, Masako TAKAMURA, Shoji KOJIMA1977 Volume 81 Issue 4 Pages 1011-1016
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of cadmium (Cd2+), zinc (Zn2+), and calcium (Ca2+) ions on the esterification of cholesterol in sonicated dispersions of lecithin-cholesterol mixtures by lecithin-cholesterol acyltransferase (LCAT) in human plasma were studied in vitro.
The acyltransferase activity was slightly stimulated at concentrations of Ca2+ ions ranging from 1×10-6M to 1×10-3M but depressed at concentrations above 1×10-4M Cd2+ ions and above 5×10-4M Zn2+ ions. The concentration of Zn2+ ions required for 50% inhibition of the acyltransferase activity was approximately twice that of Cd2+ ions. The acyltransferase inhibition caused by the addition of 1×10-3M Cd2+ or Zn2+ ions was completely overcome by the addition of 0.1% of ethylenediaminetetraacetic acid (EDTA) but not by the addition of 1×10-3M citrate, cysteine, mercaptoethanol or Ca2+ ions.
The decreased acyltransferase activity in the presence of 1×10-3M Cd2+ or Zn2+ ions increased linearly on the addition of increasing amounts of bovine serum albumin. The decreased acyltransferase activity in the presence of 1×10-3M Zn2+ (65.4 pg as Zn per ml of incubation medium) or Cd2+ (112.4 beg as Cd per ml of incubation medium) ions was restored to the control level or approximately 70% of the control level, respectively, by the addition of 40mg of bovine serum albumin per ml of the incubation medium. Thus the molar ratio of Zn2+ binding to bovine serum albumin may be greater than that of Cd2+ binding.
In the presence of Cd2+ or Zn2+ ions, the acyltransferase activity toward a dispersion with a lecithin/cholesterol molar ratio of 5.9 was decreased more than that toward a dispersion with a ratio of 1.2. This result suggests that increasing amounts of lecithin in the dispersion added as a substrate may enhance the interfering effect of Cd2+ and Zn2+ ions on the acyltransferase.View full abstractDownload PDF (438K) -
Masahiro ARIKI, Toshio FUKUI1977 Volume 81 Issue 4 Pages 1017-1024
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe action of phosphorylase b from rabbit muscle and potato phosphorylase was inhibited to various extents by several glucose analogs. Like glucose itself, all of the glucosidic oxygensubstituted analogs tested in kinetic experiments showed a nonlinear competitive inhibition for muscle phosphorylase b and a linear competitive one for potato phosphorylase. 5-Thio-D-glucose, one of the ring oxygen-substituted analogs, also inhibited the action of muscle phosphorylase b in the same manner, while the inhibition pattern of 5-amino-D-glucose (nojirimycin) was of a linear noncompetitive type. Since the conformation of 5-amino-D-glucose in aqueous solution is half-chair (Reese et al. (1971) Carbohyd. Res. 18, 381-388), the unusual kinetic behavior of the compound toward muscle phosphorylase b was supposed to be due to its halfchair conformation. In the glucosidic oxygen-substituted analogs, the affinity for both muscle phosphorylase b and potato phosphorylase decreased with decreasing order of magnitude of electronegativity of the glucosidic atom. The strong positive correlation between the affinity and the electronegativity suggests that D-glucose-1-P, the substrate, may bind to phosphorylase with the formation of a hydrogen bond between its glucosidic oxygen and a hydrogen donor of the enzyme.View full abstractDownload PDF (629K) -
Gaku ASHIBA, Shizuo WATANABE1977 Volume 81 Issue 4 Pages 1025-1030
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAccording to Arai and Watanabe (1968), γ component is a contaminant protein in preparations of skeletal muscle troponin. In the present study we sought to determine whether r component and creatine kinase [EC 2. 7. 3. 2] are identical. Both γ component and creatine kinase were prepared from chicken breast muscle and compared in four different ways (a, b, c, and d). γ Component was shown in each of these ways to be different from creatine kinase.
a) As reported by Lee and Watanabe (1970), γ component was obtained as a DEAE-cellulose flow-through fraction. No creatine kinase activity was detected in this fraction. Using the same conditions as for the r component preparation, creatine kinase preparations were all retained by DEAE-cellulose. b) In urea-polyacrylamide gel electrophoresis, γ component hardly migrated towards the anode even at pH 9.6, whereas creatine kinase migrated readily. In SDS-polyacrylamide gel electrophoresis both γ component and creatine kinase migrated similarly, but the migration rate of γ component was slightly faster than that of creatine kinase. c) Ouchterlony's immunodiffusion test with anti-creatine kinase serum of rabbit was performed, and no precipitin line was observed for γ component. d) γ Component was found to be retained by CM-cellulose, and could thus be purified further by CM-cellulose chromatography with a KCl concentration gradient. The γ component thus purified was used to obtain anti-γ component serum from rabbit, and this antiserum did not form a precipitin line with creatine kinase.View full abstractDownload PDF (1692K) -
Kazuko OHGI, Masachika IRIE1977 Volume 81 Issue 4 Pages 1031-1039
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe NBS oxidation of RNase M, which is base non-specific, was studied mainly by circular dichroism spectroscopy. It was found that tryptophan residues of RNase M were resistant to NBS at pH's from 2.5 to 6.0, but were oxidized at pH 2.0. The CD spectrum of the native RNase M gave sharp negative bands at 298 nm (band A) and 289nm (band B) and a very complex broad band around 280-265nm.
On increasing addition of NBS, band A decreased first, then band B. The decrease in band A seemed to parallel the decrease of enzymatic activity. The wavelengths of bands A and B indicated that these bands were due to tryptophan residues in RNase M. The amino acid composition of the NBS-oxidized RNase M indicated that, in the early stage of NBS oxidation, only tryptophan residues were lost and little decrease of tyrosine residues was observed.
In 8M urea, RNase M was resistant to NBS at pH's between 4.5-6.0. However, at pH 4.0, tryptophan residues of RNase M were susceptible to NBS. The mode of oxidation of RNase M was different from that in an aqueous solution, that is, band A and B decreased in parallel at the early stage of oxidation.
The changes of UV and CD spectra of RNase M in 8M urea were measured as a function of pH. A marked transition of the spectra was observed at pH 4.0. A similar experiment in aqueous solution gave a marked transition of the CD difference spectrum at pH 2.0. These results suggest that in the presence and absence of 8M urea, a conformational change in RNase M induced by decrease in pH rendered tryptophan residues in RNase M susceptible to NBS.
In the early stage of NBS oxidation, the gross structure of the enzyme was very similar to that of the native enzyme as regards the CD spectrum at 230-2 10nm, but the conformations around tyrosine residues were affected.View full abstractDownload PDF (618K) -
Masatoshi NAKANO, Yukiko SUMI, Masasumi MIYAKAWA1977 Volume 81 Issue 4 Pages 1041-1049
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat intestinal trehalase was solubilized from microvillous membrane and purified about 30-fold from the membrane fraction. The members fraction was digested with papain and then solubilized with a mixture of 0.5% Triton X-100 and 0.2% sodium deoxycholate. Solubilized supernatant was precipitated with solid ammonium sulfate, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The active fraction from the DEAE-cellulose column was subjected to polyacrylamide gel electrophoresis or ultracentrifugation analysis.
The apparent molecular weight of the trehalase was estimated to be 240, 000 by Sephadex G-200 gel filtration. Intestinal trehalase was dissociated by sodium dodecyl sulfate into fragments with a molecular weight of 30, 000. The optimum pH of the enzyme was 5.7 in phosphate buffer or histidine buffer, and shifted to 4.7 in acetate buffer. The K. value for trehalose was found to be 5.5mm in phosphate buffer and 8.0 to 11mm in acetate buffer. The Hill coefficient (n) was more than 1.0 in the case of acetate buffer. The ri values were 1.14 and 1.58 in 5mm and 500mm acetate, respectively. The n values in the presence of SH-blocking agents were larger than 1.0, and increased in the order NEM, PCMS, HgCl2. Inhibitory effects of SH-blocking agents were decreased by acetate. Thus, intestinal trehalase might undergo conformational changes in acetate.
Divalent cations such as CaCl2, MgCl2, and ZnCl2, slightly stimulated the enzyme at final concentrations of 2.5 to 5.0mm. However, the activity was not activated by sodium ions, as was the case with sucrase. SH-blocking agents and phlorizin partially inhibited the activity. Tris and SDS inhibited the activity by 80-90%. It is suggested that intestinal trehalase might play a role in sugar transport in intestinal mucosal cells.View full abstractDownload PDF (1074K) -
Toshihiko OKUDA, Shintaro SUGAI1977 Volume 81 Issue 4 Pages 1051-1056
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe transfer enthalpy Q6 of α-lactalbumin from water to aqueous guanidine hydrochloride (GuHCl) solution at pH 6.0 and the transfer enthalpy Q68 from aqueous GuHCl at pH 6.0 to pH 2.3 were measured. The dependence of Q6 on the denaturant activity indicated that (1) guanidinium ions bind to the protein in the native (N) state with a binding constant of KN=0.31, (2) the transition enthalpy from the N to the denatured (D) state in the absence of denaturant is 25.4 kcal/mol, where the binding sites (total number ΔnD, binding constant KD=1.2) were assumed to be exposed anew through the N_??_D transition. Also, from values of the transfer heat Q062 from pH 6.0 to pH 2.3 in 0.1M NaCl solution and of the calculated protonation enthalpy of the protein in the pH range of 6.0-2.3, the transition enthalpy from the N to the acid (A) state was determined to be 24±3 kcal/mol in the absence of denaturant.
The van't Hoff enthalpies of both the N_??_A and N_??_D transitions of the protein determined from CD and difference spectra measurements were compared with the calorimetric values. Each transition in the three-state transition processes of the protein was concluded to satisfy the two-state hypothesis of Lumry et al. in the absence of the denaturant.
On the other hand, ΔnD and ΔnD (the total numbers of binding sites reexposed through the N_??_D and N_??_A transitions, respectively) were estimated to be about 23 and 10, respectively, in fair agreement with the values previously determined from spectroscopic measurements.
The calorimetric transition enthalpies and the binding parameters for α-lactalbumin obtained in the present study are compared with those for lysozyme [EC 3. 2. 1. 17].View full abstractDownload PDF (445K) -
IX. Further Studies on the Spectroscopic Characteristics of the Enzyme-FMN IntermediatesNaoki ASHIZAWA, Takao NAKAMURA, Takahide WATANABE1977 Volume 81 Issue 4 Pages 1057-1062
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe absorption and fluorometric changes of the reaction mixture of luciferase-FMNH2 complex with 02 were re-examined. Rapid formation (k2(app)=2.0 s-1 at [O2]=120μM) of an intermediate with a single absorption maximum at 380nm within the range of 350-550nm, and a weak fluorescence at 520nm (≤10% of that of FMN when excited at 380nm) was observed. The absorption and fluorescence spectra and decay rate of the intermediate were estimated by simulation using an analog computer. The decay rate (0.27s-1 at 20°C) was in agreement with that of an obligatory intermediate of the luminescent reaction previously determined by measuring aldehyde-initiated luminescence. The process of decay of X1 to FMN involved another intermediate X1' with spectroscopic characteristics rather similar to those of FMN.View full abstractDownload PDF (474K) -
Yoshio TSUJITA, Akira ENDO1977 Volume 81 Issue 4 Pages 1063-1070
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical and physical properties of the carbohydrate moiety of acid protease Al [EC 2. 4. 23. 6], one of the two acid proteases produced by Aspergillus oryzae grown on solid bran media, have been studied. The enzyme was separated into two active components (A1a and A1b) by isoelectric focusing; both contained approximately 50% carbohydrate. The carbohydrate portion of the major component Al. was composed of galactose, glucose, and mannose, but the minor component A1b contained glucose as the predominant carbohydrate, with smaller amounts of galactose, fructose, xylose and mannose.
The absence of a covalent linkage between the polysaccharide and the protein in acid protease A1 was indicated, although these two moieties were tightly associated with each other. This conclusion was based on the following observations: (a) the polysaccharides obtained after proteolytic digestion contained no detectable amino acids; (b) no hexosamines were detected in acid protease A1; (c) the polysaccharides were not separable from the protein by a variety of procedures, including treatment with cold 15% trichloroacetic acid, 8M urea, 6M guanidine hydrochloride, and 0.1 N NaOH (at 25°C for 18 h). However, when treated with 50% acetic acid at 37°C for 48 h, the polysaccharides were completely dissociated from the protein; and (d) of several glycoproteins tested, containing an N-glycosyl or O-glycosyl linkage between the protein and carbohydrate moieties, none liberated carbohydrate residues upon treatment with 50% acetic acid.View full abstractDownload PDF (655K) -
III. Effects of Sodium Azide on the Enzyme FunctionsHiroshi KOBAYASHI, Masatomo MAEDA, Yasuhiro ANRAKU1977 Volume 81 Issue 4 Pages 1071-1077
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) Sodium azide and diphenyl phosphorazidate (DPPA) inhibited purified membrane-bound ATPase [coupling factor of oxidative phosphorylation; EC 3. 6. 1. 3] of Escherichia coli noncompetitively with K1 values of 39 and 51 μM, respectively.
2) Sodium azide and DPPA inhibited the activity of ATPase bound to the membrane as effectively as that of the purified enzyme.
3) The effects of sodium azide on succinate-dependent ATP synthesis, P1-ATP exchange, and ATP hydrolysis reactions by the membrane vesicles were compared under the same conditions. At concentrations below 1.0mM, sodium azide inhibited ATP hydrolysis, but P1-ATP exchange and ATP synthesis were almost unaffected. At 10mM sodium azide, both P1-ATP exchange and ATP synthesis reactions were completely inhibited, probably because at this concentration, sodium azide acted as a proton-conducting uncoupler.View full abstractDownload PDF (530K) -
Eisuke HANADA, Kunio KONNO, Shizuo HANDA, Tamio YAMAKAWA1977 Volume 81 Issue 4 Pages 1079-1083
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycolipids in rat granulomas were examined every week for six weeks after agar implantation. Furthermore, tentative identification of the glycolipids in rat granulomas was carried out by thin layer chromatography and sugar analysis by gas-liquid chromatography.
The amounts of neutral glycolipid and ganglioside per gram wet weight of granuloma were the lowest in the first and second weeks, increased rapidly to the maximal level in the fourth week and then decreased gradually thereafter. Changes in the contents of these glycolipids were similar to those of phospholipid, but it was noticeable that the rate of increase of ganglioside content was significantly higher than those of the other two.
An analysis of glycolipids in the granulomas by thin layer chromatography and sugar analysis by gas-liquid chromatography showed that the principal glycolipids were ceramide tetrasaccharide and hematoside. Four other neutral glycolipids were also detected: ceramide monohexoside, ceramide dihexoside, ceramide trihexoside, and an unidentified glycolipid (glycolipid X). Glycolipid X is considered to be a new glycolipid, the carbohydrate moiety of which contains glucose, galactose, N-acetylgalactosamine, and fucose in a molar ratio of 1:3:1:1. Five other gangliosides were also found.
Glycolipid patterns on thin layer chromatography remained virtually unchanged during 6 weeks except that the band of glycolipid X was the most prominent in the first week but became gradually less prominent and was not detectable after the fifth week.View full abstractDownload PDF (1564K) -
Teruo NISHIDA, Shingo KUME, Masayuki SAITO, Masami SUDA1977 Volume 81 Issue 4 Pages 1085-1090
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and sensitive method for the determination of threonine in rat blood plasma using aldehyde dehydrogenase after oxidation with periodate was developed. By the present method, threonine could be completely discriminated from serine and determined at the nanomole level. The amount of threonine in rat blood plasma obtained by the present method coincided well with the value determined on an amino acid analyzer.View full abstractDownload PDF (396K) -
Kazuei IGARASHI, Yoko AOKI, Seiyu HIROSE1977 Volume 81 Issue 4 Pages 1091-1096
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of polyamines and magnesium to synthetic polynucleotides has been studied by gel filtration on a Sephadex G-50 column. Among the single-stranded polynucleotides examined [poly (A), poly (C), and poly (U)], polyamines were found to bind to poly (C) and poly (U) preferentially, while the binding of Mg2+ was greatest with poly (A). Spermine bound to poly (U) was displaced completely by NH4+ but incompletely by Mg2+, while Mg2+ bound to poly (A) was displaced completely by spermine but incompletely by NH4+. The optimal pH for the binding of spermine to poly (U) was found to be about 7.9, while Mg2+ could bind to poly(A) over a broad pH range (7.1-8.7).View full abstractDownload PDF (371K) -
Keizo INOUE, Kenji SUZUKI, Shoshichi NOJIMA1977 Volume 81 Issue 4 Pages 1097-1106
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMixtures of lysolecithin with various phospholipids were studied by electron microscopy using negative staining. Mixtures of dipalmitoyllecithin and lysolecithin produced disc-shaped structures which were stacked in aggregates with a 6.0-6.4nm repeat. The disc were 10-50nm in diamter. The disc-shaped structures were best observed in equimolar mixtures of dipalmitoyllecithin and lysolecithin. When dipalmitoyllecithin was replaced by dimyristoyllecithin, the structures were rather different from those observed in the system containing dipalmitoyllecithin; a cylindrical micellar phase was predominant. Equimolar mixtures of egg lecithin and lysolecithin formed the more usual smectic, concentric lamellae (liposomes) and elongated rod-like micelles which might be bimolecular fragments of spherules. The radius of the rod-like micelles was about 6 nm. Structures of rod-like micelles were observed more frequently in the samples after incubation at room temperature and then further incubation at 0°C. Equimolar mixtures of didecanoyllecithin and lysolecithin produced large amounts of elongated rod-like micelles. Beef brain sphingoymyelin showed disc-shaped structures when mixed with lysoleci-thin. Incorporation of cholesterol into the mixtures of dipalmitoyllecithin and lysolecithin changed the morphological structure; the size of the disc became larger and eventually liposomes were formed with an increase of cholesterol content. The structures observed in mixtures of dipalmitoyllecithin or sphingomyelin and lysolecithin closely resembled those observed in complexes of apolipoprotein and lipid.View full abstractDownload PDF (4232K) -
Tairo OSHIMA, Hideji ARAKAWA, Misako BABA1977 Volume 81 Issue 4 Pages 1107-1113
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcidophilic, thermophilic bacteria were isolated from Japanese acidic hot springs. They were spore-forming rods, identified as Bacillus acidocaldarius. DNA extracted from these acidothermophiles showed no abnormality in chemical structure; it was instantly denatured and gradually decomposed giving rise to apurinic acid in a hot acid environment milder than the optimal conditions for the growth of the acido-thermophiles. Glyceraldehyde-3-phosphate dehydrogenase extracted from B. acidocaldarius was not active at pH 5 or less, and was resistant to heat at neutral but not acid pH. The intracellular pH was computed to be neutral by using dimethyl-2, 4-oxazolidinedione. When uncouplers or inhibitors of respiration were added to the cells suspended in hot acid solution, the estimated pH was not changed and glyceraldehyde-3-phosphate dehydrogenase in the cells was not denatured. These results suggest that the cytoplasm of B. acidocaldarius is a hot neutral environment, and that a pH gradient across the cell envelope can be maintained even when oxidative phosphorylation or respiration is inhibited.View full abstractDownload PDF (1092K) -
Ryoko KURIYAMA1977 Volume 81 Issue 4 Pages 1115-1125
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMarine egg tubulin could be purified from unfertilized or fertilized sea urchin and starfish eggs by the use of DEAE-Sephadex A-50 ion exchanger. About 0.2%. of the total protein in the 20, 000g supernatant of the egg extract was recovered as DEAE-Sephadex purified tubulin which assembled into microtubules upon incubation at 35°C. Applying the routine polymerizationdepolymerization procedure for tubulin purification to the initial egg tubulin preparation, almost pure egg tubulin could be obtained.
The purified egg tubulin fraction was shown by analytical centrifugation to consist of only a 6.3S component, having a molecular weight of 110, 000 as determined by gel-filtration or of 128, 000 as determined by the sedimentation-equilibrium method. Egg tubulin dimer possessed binding activities of 0.8 mol colchicine and 0.80-0.97 mol of exogenous 3H-GTP at the exchangeable site. Its α and β subunits showed the same mobilities as those of porcine brain tubulin or outer fiber tubulin of sea urchin sperm flagella on SDS-polyacrylamide gels.
When the egg tubulin fraction was warmed at 35°C, microtubules were reconstituted in parallel with the increase in viscosity. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, colchicine or SH reagents all inhibited in vitro polymerization of egg tubulin as well as inducing depolymerization of reconstituted egg microtubules. The purified egg tubulin fraction polymerized into microtubules by itself, the assembly being strikingly stimulated by the addition of exogenous nuclei fractions for polymerization.View full abstractDownload PDF (2679K) -
Hideo TAKESHIMA, Chiaki KITAO, Satoshi OMURA1977 Volume 81 Issue 4 Pages 1127-1132
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCerulenin, an inhibitor of fatty acid synthesis, specifically inhibits the biosynthesis of leucomycin, a 16-membered macrolide antibiotic, in both growing cells and resting cells of Streptomyces kitasatoensis. In growing cells, the production of leucomycin was inhibited as long as cerulenin remained in the culture. In resting cells, 50 percent inhibition was achieved with a cerulenin concentration of 1.5 μg/ml. Cells in which leucomycin synthesis was inhibited for 9 h remained capable of leucomycin synthesis upon removal of the inhibitor. Cerulenin specifically inhibits the incorporation of [14C]acetate into leucomycin but does not affect total protein or RNA synthesis. The uptake of [14C]acetate was not inhibited under conditions which completely inhibited the incorporation of acetate into leucomycin. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it can be concluded that the aglycone of leucomycin is synthesized via the polyketide pathway by condensation steps similar to those involved in fatty acid biosynthesis.View full abstractDownload PDF (403K) -
Hiroyuki ARATA, Ken-ichiro TAKAMIYA, Mitsuo NISHIMURA1977 Volume 81 Issue 4 Pages 1133-1139
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDelayed fluorescence from bacteriochlorophyll in the chromatophores of Chromatium vinosum, a photosynthetic purple sulfur bacterium, was studied in the presence of o-phenanthroline (o-phen) under intermittent illumination. Re-reduction of the photooxidized reaction center bacteriochlorophyll (P+) in the dark interval was accelerated by o-phen. This effect was attributed to the return of electrons trapped in the primary electron acceptor (A) to P+.
In the presence of o-phen, the time course of the decay of delayed fluorescence was not coincident with that of the re-reduction of P+. The delayed fluorescence was somewhat intensified at the early stage (within 30 ms) of relaxation in the dark period. Prolonged illumination (longer than 20 ms) or uncouplers such as carbonylcyanide m-chlorophenylhydrazone (CCCP) or valinomycin plus nigericin decreased the intensity of delayed fluorescence and suppressed the stimulation of delayed fluorescence at the early stage.
Delayed fluorescence from reaction center-rich subchromatophore particles decayed with a time course identical to that of the reduction of P+ and was not affected by CCCP, in the presence of o-phen.
The intensification at the early stage in the chromatophores can be interpreted in terms of charge separation between pairs of P and A, primary electron donor and acceptor molecules, oriented perpendicular to the intact chromatophore membrane, the effect decreasing in parallel with the recombination of P+ and A-.View full abstractDownload PDF (491K) -
Tetsu HOZUMI, Ken HOTTA1977 Volume 81 Issue 4 Pages 1141-1146
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.View full abstractDownload PDF (508K) -
I. Affinity Labeling of Glyceraldehyde 3-Phosphate Dehydrogenase and Myokinase with a Reactive AMP AnalogKoichi SUZUKI, Chikahiko EGUCHI, Kazutomo IMAHORI1977 Volume 81 Issue 4 Pages 1147-1154
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD) and myokinase (MK) were rapidly inactivated by a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP, under mild conditions. Complete inactivation was observed when 4 and 0.3 mol of the reagent with respect to enzyme were reacted with GPD and MK, respectively. The inactivation of both enzymes was favored at higher pH and the enzymes were protected by addition of adenine nucleotide substrate. Modified GPD or MK had no affinity for AMP-Sepharose, in contrast to the native enzymes. From these results, the inactivation of GPD and MK by the reactive AMP analog can be regarded as an affinity labeling. The possibility that the present AMP analog may be used as a general affinity labeling reagent for various adenine nucleotide-related enzymes is discussed based on the results obtained.View full abstractDownload PDF (622K) -
Hiroko TAMURA-TAKAHASHI, Nobuo UI1977 Volume 81 Issue 4 Pages 1155-1160
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour biologically active components of luteinizing hormone (LH), designated as LH I, II, III, and IV, were isolated from whale pituitary glands. These components migrated as single bands with different mobilities in disc electrophoresis, and their isoelectric points were at pH 8.1, 8.3, 8.5, and 8.7, respectively. Their amino acid and carbohydrate compositions showed close similarity. The molecular weight of whale LH was determined to be 31, 000 by sedimentation equilibrium, and no difference was detected among the four components. The molecular properties of whale LH were compared with the previously reported data for LH from other animal species.View full abstractDownload PDF (1070K) -
Yasuo KAGAWA, Toshio ARIGA1977 Volume 81 Issue 4 Pages 1161-1165
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular species of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin of thermophilic bacterium PS3 were determined by a new and simple mass chromatographic technique. The fatty acyl groups of these compounds, unlike those of usual phospholipids, were all saturated and were mixtures of even and odd numbered carbcn chains; their [M-CnH2n+1-COOCH2]+ and [M-Cn+1H2n+3+COO]+ peaks overlapped. Thus, the acetyldiglycerides derived from these phospholipids were subjected to a combination of mass chromatography using chemical ionization with NH3 and treatment of the phospholipids with phospholipase A2 [EC 3. 1. 1. 4].
These three phospholipids had similar patterns of diglycerides. Expressed as R1:R2, %, they were found to be 14:14, 0-1%, 15:14, 0-4%, 15:15, 22-32%, 16:15, 16-20%, 17:15, 28-32%, 17:16, 6-9%, 18:15, 7-13%, 17:17, 3-6%, and 18:16, 0-1%.
These results should be of significance in connection with the high stability of the biomembranes of this bacterium.View full abstractDownload PDF (328K) -
Yousuke SEYAMA, Takeshi KASAMA, Tamio YAMAKAWA, Akihiko KAWAGUCHI, Shi ...1977 Volume 81 Issue 4 Pages 1167-1173
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium aminoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained:
1) HB hydrogen of NADPH was used for p-ketoacyl reductase.
2) HB hydrogen of NADH was used for enoyl reductase.
3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon atoms.
4) Hydrogen atoms from NADPH was found on the odd-numbered methylene carbon atoms (1 hydrogen per carbon).
5) Hydrogen atoms from NADH was also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected.
6) The exchange of methylene hydrogen atoms of malonyl-CoA with protons of water was suggested by 13C NMR analysis.View full abstractDownload PDF (483K) -
Junji HASHIMOTO, Kenji TAKAHASHI1977 Volume 81 Issue 4 Pages 1175-1180
Published: April 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3. 1. 4. 8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about I residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.View full abstractDownload PDF (950K)
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