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Volume 87 (1980)
- Issue 6 Pages 1559-
- Issue 5 Pages 1261-
- Issue 4 Pages 1003-
- Issue 3 Pages 679-
- Issue 2 Pages 369-
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Volume 87, Issue 5
Displaying 1-34 of 34 articles from this issue
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Yuko YAMADA, Yoshiyuki KUCHINO, Hisayuki ISHIKURA1980 Volume 87 Issue 5 Pages 1261-1269
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInitiator methionine tRNA, tRNAMetf was purified from Bacillus subtilis W168 by the use of two column chromatographies on DEAE-Sephadex A-50 and BD-cellulose. The nucleotidesequence was determined to be pC-G-C-G-G-G-G-U-G-G-A-G-C-A-G-U-U-C-G-G-D-A-G-C-U-C-G-U-C-G-G-G-C-U-C-A-U-A-A-C-C-C-G-A-A-G-G-U-C-G-C-A-G-G-T-Ψ-C-A-A-A-U-C-C-U-G-C-C-C-C-C-G-C-A-A-C-C-AOH. This tRNAMetf exhibited a melting temperature 8°C lower than that of E. coli tRNAMetf in the presence of 0.01 M magnesium acetate.View full abstractDownload PDF (1198K) -
Kyosuke YAMAMOTO, Fumiko KAMO-YAMADA, Shuji CHO, Michihiro SUGANO1980 Volume 87 Issue 5 Pages 1271-1278
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCholesterol esterification and cholesterol ester hydrolysis in dog plasma were investigated. Esterification proceeded linearly for 60min, and the amounts of cholesterol esterified were in the range of 0.13-0.18 μmol/ml/h. No change of acyl composition had occurred in newly formed cholesterol esters during incubation. With the addition of Na taurocholate (10mM), complete inhibition of the esterifying activity and maximal activation of the hydrolase activity were observed. Approximately 50% of cholesterol esters present in plasma was hydrolyzed in 10 min of incubation, and the reaction was completed within 60 min. The maximal rate of hydrolysis was estimated to be 4.0-5.4 μmol/ml/h, and polyunsaturated esters were hydrolyzed more rapidly than saturated ones. The esterifying activity was detected in high density (HDL) and very high density lipoproteins (VHDL), while the hydrolytic activity was found only in VHDL. Each lipoprotein fraction served as a good substrate for hydrolysis, while HDL was the sole substrate for esterification. The optimal pH of the hydrolytic activity in VHDL lay in a broad range between 6.8 and 7.2 and the apparent Km was determined as 12.5×10-3mM for cholesteryl oleate.View full abstractDownload PDF (548K) -
Makio KITADA, Koki HORIKOSHI1980 Volume 87 Issue 5 Pages 1279-1284
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkalophilic Bacillus No. 8-1 and No. 10 A-2, isolated on alkaline media containing NaHCO3 and K2CO3, respectively, required Na+ specifically for the uptake of α-aminoisobutyric acid (AIB) into the cells. The K+ added to the uptake medium inhibited the accumulation of AIB in the cells of Bacillus No. 8-1, acting as a competitive inhibitor of the system. Sodium ion prevented the release of AIB from pre-loaded cells. Both potassium and lithium ions had the effect of retaining the AIB pool.
The Vmax value for transport increased about five-fold when the pH value of medium was raised from 7 to 9, whereas the Km value decreased with the increase of concentration of sodium ion in the medium of pH 7 or 9.
Sodium ion gradient across the membrane caused transient uptake of AIB by membrane vesicles of Bacillus No. 8-1.View full abstractDownload PDF (444K) -
Takashi SUZUKI, Rensuke GOTO, Osamu TAMEMASA1980 Volume 87 Issue 5 Pages 1285-1291
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNonhistone chromasomal proteins (NHP) of Ehrlich ascites tumor cells were fractionated into four subfractions by successive extractions with 0.35 M, 0.45 M, and 0.60 M NaCI, and finally 2.0 M NaCl+5.0 M urea. The CD spectra and transcriptional activities of complexes reconstituted from DNA, histone, and each of these four NHP subtractions obtained from Ehrlich tumor cells were measured in order to identify the effect of each NHP subfraction. The positive ellipticity in the CD of the DNA-histone complex was considerably increased when reconstituted with 0.35 M- or 0.45 M-NHP. The 0.35 M- and 0.45 M-NHP also increased the transcriptional activity of DNA-histone complex. The activities of DNA-histone-0.35 M NHP complex increased progressively with increase in the 0.35 M NHP/DNA ratio. These results suggest that there is a correlation between the increase in transcriptional activity of DNA-histone complexes with 0.35 M- or 0.45 M-NHP and the increase in the percentage of DNA in the B conformation in complexes.View full abstractDownload PDF (2171K) -
Yasuo OGAWA, Hikaru HARAFUJI, Nagomi KUREBAYASHI1980 Volume 87 Issue 5 Pages 1293-1303
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The characteristics of four calcium indicators, murexide (MX), tetramethylmurexide (TMX), arsenazo III (Az) and antipyrylazo III (Ap), were examined to assess their applicability for biological experiments.
2. Az has the following serious disadvantages, although it has a high sensitivity to Ca: (i) nonlinearity of absorbance change against the concentration of Ca due to a change in the structural composition of its complex (2:1 complex to 1:1 complex) in the range below 10 μM Ca2+, and due to the high apparent binding constant to Ca at 20 μM or higher Ca2+; (ii) severe interference by Mg2+; (iii) rather low association and dissociation rate constants with Ca which are not sufficient under conditions where Ca2+ to be determined in the system cannot be disturbed by Az; (iv) inhibitory effect on Ca uptake by bullfrog fragmented sarcoplasmic reticulum.
3. Ap has similar disadvantages, although Ap reacts with Ca faster than Az. (i) Ap suffers from interference by Mg2+ even though the effect of Mg2+ can be minimized at the expense of reduced sensitivity. (ii) Plots of absorbance change versus the concentration of Ca deviate from linearity around 20 μM Ca2+ or higher. (iii) Ap is not very sensitive at concentrations which would not disturb Ca2+ in the system to be determined.
4. TMX is twice as sensitive as MX. However, TMX may be more permeable through membranes than MX in the presence of ATP.
5. MX has no disadvantages except for its low sensitivity to Ca2+View full abstractDownload PDF (1984K) -
Hikaru HARAFUJI, Yasuo OGAWA1980 Volume 87 Issue 5 Pages 1305-1312
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The apparent binding constant (Kapp(Ca-G)) for GEDTA (ethylene glycol bis(β-aminoethyl ether)-N, N, N', N'-tetraacetic acid, EGTA) to calcium was determined under conditions of biological significance in the presence of various kinds of pH-buffering agents, using murexide or tetramethylmurexide as a Ca indicator.
2. The value of Kapp(Ca-G) at pH 6.80 was 1.0×106M-1 at an ionic strength of 0.114 at 20°C, irrespective of the type of pH-buffering ions. This value is similar to that of Allen, Blinks and Prendergast (1977) (Science 196, 996-998), but still half that calculated from the results of Schwarzenbach, Senn and Anderegg (1957) (Helv. Chim. Acta 40, 1886-1900).
3. The value of Kapp(Ca-G) varied according to the following equation as the ionic strength (I) was varied from 0.039 to 0.264:
log Kapp(Ca-G)=6.460-[2_??_I/(1+_??_I)-0.4×I] (pH 6.80, 20°C)
4. The discrepancy between the present results and previous ones (Ogawa, Y. (1968) J. Biochem. 64, 255-257) may have been due to inadequate regulation of the temperature of the reaction medium in the previous determinations, during which an increase in the temperature of the solution may have occurred.
An increase of temperature causes a decrease in the pH of the solution in the presence of histidine, imidazole or Tris-maleate, but causes very little change of pH in the presence of phosphate or maleate.
5. The association rate constant for GEDTA with calcium was determined by the stoppedflow method in solutions containing 100mM KCl and 20mM pH-buffering ions at 20°C: the values obtained were 1.4×106M-1s-1 in the presence of MOPS-KOH at pH 6.80; 3.0×106M-1s-1 with imidazole at pH 6.80; 1.0×106M-1s-1 with Tris-maleate at pH 6.80.View full abstractDownload PDF (1286K) -
Comparison with Calmodulins from Rabbit Skeletal Muscle and Pig BrainMichio YAZAWA, Mami SAKUMA, Koichi YAGI1980 Volume 87 Issue 5 Pages 1313-1320
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInvertebrate calmodulins of the sea anemone and scallop muscle were isolated and their properties were compared with those of vertebrate calmodulins from rabbit muscle and pig brain. The molecular weights estimated by SDS-polyacrylamide gel electrophoresis were similar to the molecular weight (16, 500) of the vertebrate calmodulins. Every calmodulin contained 1 mol each of trimethyllysine and histidine, and high contents of acidic amino acids. The marine invertebrate calmodulins contained only one tyrosine in contrast to two tyrosines in the vertebrate ones. As a result, the UV absorption spectra were clearly different. The Ca2+-induced difference UV absorption spectra of the invertebrate calmodulins were indistinguishable from those of the vertebrate ones in spite of the difference in tyrosine contents. In tryptic peptide maps of invertebrate calmodulins, a few spots different from those of vertebrate calmodulins were observed in the basic and acidic peptide regions. The calmodulins of invertebrate muscles and that of rabbit skeletal muscle were almost indistinguishable in terms of the activation profile of rabbit skeletal myosin light chain kinase.View full abstractDownload PDF (2094K) -
Katsuhisa FUJISHIRO, Yukihiro ASNADA, Hideshi TANAKA, Nobuhiko KATUNUM ...1980 Volume 87 Issue 5 Pages 1321-1326
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtease B [EC 3. 4. 22. 9] was purified from baker's yeast by plasmolysis of yeast, acid activation, acid precipitation, and column chromatographies on QAE-Sephadex, SP-Sephadex, D-tryptophan methyl ester-Sepharose 4B and Sephadex G-100. The purified enzyme was inhibited by phenylmethylsulfonyl fluoride and sulfhydryl-blocking reagents. Chymostatin and antipain at extremely low concentrations (1 μM) inhibited the protease B. The effects of the enzyme on various yeast enzymes were examined by measuring their inactivation. The enzyme inactivated 6-phosphogluconate dehydrogenase [EC 1. 1. 1. 44] and uricase [EC 1. 7. 3. 3], but not malate dehydrogenase [EC 1. 1. 1. 37], alcohol dehydrogenase [EC 1. 1. 1. 1], glutamate dehydrogenase [EC 1. 4. 1. 3], glucose-6-phosphate dehydrogenase [EC 1. 1. 1. 49] or hexokinase [EC 2. 7. 1. 1].View full abstractDownload PDF (441K) -
Masaru KAWAMURA, Toshiko OHTA, Kei NAGANO1980 Volume 87 Issue 5 Pages 1327-1333
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA high concentration (0.5 M) of a reducing agent such as dithiothreitol or 2-mercaptoethanol inactivated both crude (microsomal) and purified preparations of Na+, K+-ATPase only in the presence of detergent. The concentration of SDS needed for inactivation by a reducing agent was low (around 0.3mg/ml), and was similar to that required for activation in the absence of reduction. The subunits of the enzyme after inactivation with a reducing agent were not sedimented by centrifugation at 100, 000×g for 1 h. Electron microscopic observation of the inactivated enzyme revealed that membranous structures were dominant in the pellet obtained by centrifugation, whereas many particles were present in the supernatant.
These results suggest that Na+, K+-ATPase was inactivated by reduction of disulfide bond (s) embedded within the lipid bilayer in the presence of detergent, and that the subunits of the resulting enzyme were no longer bound to the membrane.View full abstractDownload PDF (2662K) -
Teruhiro TAKABE, Satsuki NIWA, Hiroshi ISHIKAWA1980 Volume 87 Issue 5 Pages 1335-1339
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe autoreduction of spinach plastocyanin has been studied by fluorescence, absorption, and paramagnetic resonance spectroscopy. Decolorization of oxidized plastocyanin, due to the autoreduction of plastocyanin, occurred rapidly at alkaline pH (half-life 20min at pH 10.2). Its rate also increased with increasing ionic strength. In the presence of a hydrophobic fluorescent probe such as 2-p-toluidino-naphthalene-6-sulfonate (TNS), the fluorescence intensity of TNS due to binding to oxidized plastocyanin gradually increased following the autoreduction of plastocyanin, which suggests that copper is autoreduced in parallel with the exposure of a hydrophobic site (probably near Cys 84) to the solvent. The EPR spectra also supported the change of coordination geometry of copper. The reduction rate of plastocyanin by ferrocyanide at alkaline pH was almost the same as that at neutral pH, and TNS did not alter the exogenous reduction rate even at alkaline pH. These results suggest that exogenous and endogenous reduction take place independently at different reduction sites in spinach plastocyanin.View full abstractDownload PDF (391K) -
Michinori MIYAHARA, Koichiro KISHI, Haruhiko NODA1980 Volume 87 Issue 5 Pages 1341-1345
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSF-Protein has an amino acid composition distinctively different from those of myofibrillar proteins so far reported to be of similar chain weight: M-protein component II, α-actinin, and AMP deaminase. Its molecular weight was estimated to be 121, 000 by sedimentation equilibrium in 0.3M KCl, 10mM potassium phosphate, pH 6.5. Its binding to myosin was inhibited by C-protein. It reduced the effect of C-protein on the assembly reaction of myosin in vitro.View full abstractDownload PDF (332K) -
Masao HAYASHI, Kazuo OHNISHI, Kazuko HAYASHI1980 Volume 87 Issue 5 Pages 1347-1355
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurified tubulin from porcine brain formed a dense precipitate at 37°C with muscle myosin filaments from rabbit skeletal muscle; this effect was greater than that with partially purified tubulin. ATP or GTP, which prevented the myosin filaments from precipitating, inhibited the formation of the dense precipitate, but did not dissociate the dense precipitate once formed.
The dense precipitate was found by thin-section electron microscopy to be composed of side-by-side aggregates of myosin filaments whose projections might be decorated by tubulin. The decoration was also seen by negative-stain electron microscopy.
The binding of tubulin to myosin filaments decreased the Mg2+- and Ca2+-GTPase activity of the myosin by about half, but did not affect either Mg2+- or Ca2+-ATPase activity. The binding ratio of tubulin to myosin in the presence of 5 mM MgCl2 was 2.2 mol/mol using purified tubulin and 1.8 mol/mol using partially purified tubulin. Five mM ATP and GTP in the presence of 5 mM MgCl2 decreased the tubulin binding by 1.6-2.0 and 1.1-1.3 mol/mol, respectively, when added before an encounter of tubulin with myosin filaments, but did not cause any decrease when added after such an encounter.View full abstractDownload PDF (3146K) -
Hidenori YOSHINO, Osamu MINARI1980 Volume 87 Issue 5 Pages 1357-1363
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of 1-anilino-8-naphthalene sulfonate to human serum high density apolipoprotein and to apoA-I and apoA-II which are major protein components of high density apolipoprotein was measured by a fluorometric method. The maximum number of 1-anilino-8-naphthalene sulfonate bound to high density apolipoprotein was about 950 μmol per g of protein, and those to apoA-I and apoA-II were 27 and 13 mol per mol of protein (these values correspond to 964 for apoA-I and 765 for apoA-II in μmol per g of protein), respectively. After preincubation of apolipoprotein with lysolecithin (less than 10 μM), the binding affinities for the dye increased twice or more with all the proteins, but the maximum binding numbers changed little. With apoA-I, two mol of the dye were bound with high affinity and lysolecithin did not compete with the dye for the binding sites.
The fluorescence intensity of tryptophanyl residues of apoA-I was changed by incubation with lysolecithin at concentrations lower than the critical micellar concentration.
These results indicate that the binding of lysolecithin molecules to apoA-I or other proteins makes the protein conformation more suitable for the binding of hydrophobic ligands.View full abstractDownload PDF (532K) -
V. Possible Involvement of Four Species of the Methyl-Accepting Chemotaxis Protein in Chemotaxis of Escherichia coliOsamu KOIWAI, Shinsei MINOSHIMA, Hiroshi HAYASHI1980 Volume 87 Issue 5 Pages 1365-1370
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChemotactic Escherichia coli contains five major methyl-accepting proteins. Three of them were identified as the product of tsr gene, tar gene and peptide elongation factor Tu. Electrophoretic analysis of sulfur-labeled proteins and methyl-labeled proteins from trg mutants, which lost the ability of chemotaxis only towards ribose, galactose and their analogs, showed that the product of trg gene was another methyl-accepting protein i.e. a methyl-accepting chemotaxis protein for ribose and galactose (trg-MCP). The last methylatable protein, named as MCP-IV, seems to be involved in chemosensory transduction (accompanying paper). Thus, it is possible that chemosensory transduction in E. coli involves four species of MCP, although no genetic evidence for MCP-IV has yet been found. A hypothesis relating a change in the methylation of MCP with a movement of ions is presented.View full abstractDownload PDF (2022K) -
VI. Effect of cheX Mutation on the Methylation of Methyl-Accepting Chemotaxis Protein of Escherichia coliShinsei MINOSHIMA, Hiroshi HAYASHI1980 Volume 87 Issue 5 Pages 1371-1377
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane from Escherichia coli cheX mutants failed to accept the methyl-moiety when incubated with a cytoplasm of wild type bacteria. The possibility of reduced production of methyl-accepting chemotaxis protein (MCP) as a result of the effect of cheX mutation was considered. However, analysis of sulfur labeled proteins from cheX mutants by two-dimensional gel electrophoresis showed that the proportion of non-esterified MCP increased in cheX mutants while the total amount of MCP was not seriously affected. Thus the decreased methylation of MCP in cheX mutants, as detected by the methyl-labeling experiment, is not caused by an effect of cheX mutation on the production of MCP back-bone protein but by a defect in the device for methylation. The presence of four species of MCP's whose extent of methylation was affected by cheX mutation as well as cheB mutation was also shown by this experiment.View full abstractDownload PDF (1940K) -
Issei OHTSUKA, Michiyuki KASAI, Ikuo KIMURA, Hirofumi ONISHI, Shizuo W ...1980 Volume 87 Issue 5 Pages 1379-1385
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree different reactions are known to occur in a combined system of skeletal actin, gizzard myosin, and gizzard native tropomyosin. They were studied as functions of ATP concentration.
(a) At around 1 μm ATP in the presence of an ATP-regenerating system, two of the three reactions, superprecipitation and ATPase reaction, occurred independently of calcium and were not accompanied by the third reaction, phosphorylation of myosin light chains. (b) Relatively high concentrations of ATP were required for calcium-dependent phosphorylation and for calcium-activated ATPase reaction. It is suggested that the apparent Km value for myosin light-chain kinase and that for acto-phosphorylated myosin-ATPase are approximately 10-4.0 M and 10-5.5 M, respectively. (c) The calcium-dependent phosphorylation and the calcium-activated ATPase reaction were closely coupled, but they were only indirectly coupled with superprecipitation. (d) In some of the responses to change in ATP concentration, (a)-(c), skeletal acto-gizzard myosin was similar to skeletal acto-skeletal myosin. It was also similar in that sulfhydryl groups of myosin are involved in the calcium regulation of actomyosin-ATPase and its superprecipitation in both cases.View full abstractDownload PDF (477K) -
Kenji OEDA, Yoshimichi NAKATSU, Mutsuo SEKIGUCHI1980 Volume 87 Issue 5 Pages 1387-1394
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTreatment of ØX174 replicative form (RF) DNA, pre-exposed to ultraviolet light, with T4 endonuclease V led to a marked increase of infectivity of the RF when the activity was assayed on CaCl2-treated cells of Escherichia coli strain defective in uvrA gene. The reaction was specific and the extent of the reactivation was proportional to the concentration of the enzyme. Based on this finding, we developed a procedure to assay endonuclease activities specific for ultraviolet-damaged DNA, that might be involved in the incision step of excision repair of pyrimidine dimers. To find conditions suitable for accurate and rapid assays, we examined conditions affecting transfection with ØX174 RF The maximum transfection was achieved when more than 2×108 CaCl2-treated cells, which had been prepared from bacteria harvested during the early or mid-logarithmic phase of growth in L broth, were incubated with the DNA at 0°C for 20 min in 50 mM CaCl2. Incubation of the cell-DNA mixture at 37°C decreased the transfection efficiency to about 30% of the optimal level; thus, heat shock, a step regarded as necessary in the conventional “CaCl2 methods” for transfection and transformation, was eliminated. The CaCl2-treated cells remained viable and competent after storage at -20°C in a solution containing 15% glycerol. By using the procedure thus established, repair endonuclease activities in crude extracts of T4-infected E. coli and of Micrococcus luteus were determined. The procedure should be of use in assaying and purifying repair enzymes of other organisms.View full abstractDownload PDF (574K) -
Subunit Structure Composed of Non-Identical Polypeptides and Pyridoxal 5'-Phosphate-Binding SubunitToshiharu YAGI, Haruo MISONO, Norio KURIHARA, Tatsuo YAMAMOTO, Kenji S ...1980 Volume 87 Issue 5 Pages 1395-1402
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Lysine: 2-oxoglutarate 6-aminotransferase from Flavobacterium lutescence (=Achromobacter liquidum)2 has been shown to be composed of one each of four non-identical subunits, A, B1, B2, and C. The subunits were isolated by gel filtration, and DEAE-cellulose chromatography in the presence of 8M urea. Their molecular weights were determined by ultracentrifugation, gel electrophoresis and gel filtration: subunit A 24, 000; Bl 28, 000; B2 28, 000; C 45, 000. These subunits were all different in amino acid composition. Of the two molecules of bound pyridoxal 5'-phosphate, the one which absorbs at 415 nm is bound to subunit B2 and participates in the catalytic action of the enzyme.View full abstractDownload PDF (2717K) -
Motoharu HAYASHI, Kiyoshi OSHIMA1980 Volume 87 Issue 5 Pages 1403-1411
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAminotripeptidase [EC 3. 4. 11. 4] was purified from monkey brain by a five-step procedure comprising extraction from brain homogenate, ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography, and Sephadex G-200 gel filtration. A purification of 1, 100-fold over the homogenate was achieved and the yield was 12%. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis at pH 8.9. The amino acid composition of the enzyme resembled that of the pig kidney enzyme. The molecular weight of the enzyme was estimated to be about 65, 000 by gel filtration on Sephadex G-200 and 70, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH optimum for L-leucyl-glycyl-glycine was about pH 7.5. The enzyme hydrolyzed only tripeptides to yield the NH2-terminal residues as free amino acids and the residual dipeptides. The enzyme did not show activities of arylamidase or carboxypeptidases A and B. The enzyme was inhibited by PCMB, o-phenanthroline, and bestatin. The inhibition by bestatin was competitive and the K1 value was calculated to be 5×10-7 M.View full abstractDownload PDF (1239K) -
Michinori MIYAHARA, Haruhiko NODA1980 Volume 87 Issue 5 Pages 1413-1420
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of C-protein on the assembly reaction of myosin was studied by flow birefringence, electron microscopy, and ultracentrifugation. Myosin filaments were formed by dilution to a lower ionic strength. Thinner filaments of 70-110 Å in diameter were formed in the presence of C-protein. When dilution was effected by moderately slow dilution (dilution time of 0.5-2min) or by stepwise dilution, C-protein favored the formation of longer filaments. When dilution was effected by even slower dilution (dilution time above 2min), C-protein favored the formation of shorter filaments. Longer filaments formed by slow dilution incorporated more C-protein than shorter ones formed by faster dilution. Addition of C-protein to a solution of myosin filaments caused association of the filaments into longer filaments. The elongation effect was slower and stronger for longer filaments.View full abstractDownload PDF (1355K) -
Takuya KATAGIRI, Isao ADACHI, Tadao TERAO, Toshiaki OSAWA1980 Volume 87 Issue 5 Pages 1421-1430
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe existence of specific binding proteins for α-casein, a well-known potent chemoattractant, on guinea pig peritoneal macrophages was demonstrated. Binding of 3H-α-casein to macrophages was found to be specific and reversible. Its association constant and the number of binding sites were 5.0×10-6 M and 7.5×105 per cell, respectively. The presence of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), another chemoattractant, at a chemotactically active concentration (10-11 to 10-6 M) reduced this binding significantly. Pretreatment of macrophages with proteases such as trypsin and chymotrypsin decreased the number of binding sites for α-casein and the chemotactic response to α-casein.
α-Casein binding proteins were isolated from radiolabeled macrophage surface membranes by affinity chromatography on a column of α-casein-Sepharose 4B. SDS-polyacrylamide gel electrophoresis of the isolated proteins revealed three radioactive bands; two (MW 130, 000 and 65, 000) were glycoproteins, and the other (MW 100, 000) was a protein which contained little carbohydrate.
These three proteins were also isolated from the same source by affinity chromatography using a column of fMet-Leu-Phe-AH-Sepharose. This result, together with the observation that fMet-Leu-Phe inhibited the binding of α-casein to macrophages, indicated that α-casein and the tripeptide have at least partly common binding sites on guinea pig macrophages.
Some of the proteins obtained in this work may constitute chemotactic receptors on the macrophage membrane.View full abstractDownload PDF (819K) -
Katsumi MATSUURA, Ken-ichiro TAKAMIYA, Shigeru ITOH, Mitsuo NISHIMURA1980 Volume 87 Issue 5 Pages 1431-1437
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe characteristics of the salt and pH dependences of the redox levels of cytochrome c2 and reaction center bacteriochlorophyll were studied in chromatophores from Rhodopseudomonas sphaeroides. They could be explained in terms of the difference of redox potential in the membrane from that in the bulk aqueous phase due to the electrostatic potential difference arising from charges fixed on the membrane surface. The midpoint potentials (Em) became lower when the surface potential (the electrostatic potential at the surface with reference to the bulk aqueous phase) had large negative values at lower salt concentrations at neutral pH, as predicted by the Gouy-Chapman theory.
The rate of oxidation of cytochrome c2 in chromatophores by ferricyanide also depended on salt and pH levels. The rate was low at low salt concentrations, probably because of the lower surface concentration of ferricyanide compared with the bulk concentration, due to the surface potential.View full abstractDownload PDF (497K) -
Katsuya GOMI, Teruhiko BEPPU, Kei ARIMA1980 Volume 87 Issue 5 Pages 1439-1448
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo novel enzymes, NAD+-linked L-alloisocitrate (erythro-Ls-isocitrate) dehydrogenase and oxalosuccinate decarboxylase, were found and purified from a strain of Pseudomonas isolated as an L-alloisocitrate utilizing bacterium. The former enzyme catalyzes a reversible oxidationreduction between L-alloisocitrate and oxalosuccinate which favors oxalosuccinate reduction. The latter enzyme catalyzes rapid decarboxylation of oxalosuccinate to α-ketoglutarate and CO2. Both enzymes require no metals for their activities. Complete oxidative decarboxylation of L-alloisocitrate to α-ketoglutarate occurs as a result of the sequential reactions catalyzed by these two enzymes. L-Alloisocitrate induces both enzymes in the growing pseudomonad.View full abstractDownload PDF (1154K) -
I. H2O2 and O2- Generation by Rabbit Alveolar MacrophagesTeruhide YAMAGUCHI, Katsuko KAKINUMA, Mizuho KANEDA, Kokichi SHIMADA1980 Volume 87 Issue 5 Pages 1449-1455
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe oxidative metabolism of rabbit alveolar macrophages (A-MØ) was compared with that of rabbit polymorphonuclear leukocytes (PMN) with respect to H2O2 generation by intact cells or subcellular fractions. Rabbit PMN exhibited an increase in the oxygen uptake and a marked release of H2O2 upon addition of heat-killed E. coli in the presence and absence of opsonin. However, rabbit A-MØ exhibited an increase in the oxygen uptake upon addition of E. coli only in the presence of anti-E. coli serum as an opsonin, whereas a very small amount of H2O2 release was observed during ingestion of the opsonized E. coli. The generation of O2 and H2O2 by a granule-rich fraction isolated from phagocytosing PMN was larger than that by a similar fraction isolated from resting PMN. However, there was no significant difference in O2- and H2O2 generation by the granule fractions between phagocytosing and resting A-MØ in the presence of either NADH or NADPH. In contrast to the granule fraction of rabbit PMN, the O2- and H2O2 generating activities in the A-MØ granule fraction were higher in the presence of NADH than in the presence of NADPH. The rates of NADH and NADPH oxidation by both A-MOslash; and PMN granule fractions were measured with and without addition of Mn2+ to the assay medium. The effect of Mn2+ on the NAD (P) H oxidase was found to differ between rabbit A-MOslash; and PMN.View full abstractDownload PDF (583K) -
Toshihiro SUGIYAMA, Nobuo MIKI, Toshio YAMANO1980 Volume 87 Issue 5 Pages 1457-1467
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA reconstituted system containing a form of cytochrome P-450, cytochrome b5, NADPH-cytochrome P-450 reductase, and NADH-cytochrome b5 reductase, all purified from rabbit liver microsomes, could catalyze O-demethylation of p-nitroanisole in the presence of both NADPH and NADH. Omission of either cytochrome P-450 or cytochrome b5 from the system led to complete loss of the activity. The reconstituted activity was sensitive to carbon monoxide, metyrapone, phenyl isocyanide, and cyanide, indicating that the cytochrome P-450 used is cyanide-sensitive and is involved in the catalytic process. The maximal demethylase activity was attained when the system contained cytochrome P-450 and cytochrome b5 at a 1:1 molar ratio. Trypsin digestion of cytochrome b5 abolished the capacity of this cytochrome to reconstitute the demethylase activity. These results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of cytochrome b5 and that the second electron needed for the demethylation may be donated to the cytochrome P-450 only by way of cytochrome b5View full abstractDownload PDF (859K) -
Observation of an Intermediary Stable Charge Transfer ComplexRetsu MIURA, Kiyoshi SHIGA, Yoshihiro MIYAKE, Hiroshi WATARI, Toshio Y ...1980 Volume 87 Issue 5 Pages 1469-1481
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of D-amino acid oxidase [EC 1. 4. 3. 3] (DAO) from porcine kidney with β-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-ECNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of β-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as α-amino-β-cyanoacrylate and the charge transfer complex as the complex of α-amino-β-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present findings is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-l enzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.View full abstractDownload PDF (826K) -
The Effects of Enzyme ConcentrationCarlos GUTIERREZ MERINO, Francisco GARCIA BLANCO, Miguel POCOVI, Marga ...1980 Volume 87 Issue 5 Pages 1483-1490
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enthalpies of binding of AMP to phosphorylase b have been measured as a function of enzyme concentration in glycylglycine buffer, pH 6.9. The results show how a conformational transition, which takes place in the concentration range of 1.7 to 2.5mg/ml of phosphorylase b, affects the enthalpies of the two binding sites per monomer for the allosteric activator AMP. The enthalpies of the AMP interaction with its higher and lower affinity binding sites are -220 and -640 kJ (mol monomer)-1 at an enzyme concentration of 1mg/ml, and -120 and -360 kJ (mol monomer)-1 at 2.7mg/ml. The conformational transition of phosphorylase b alters the reactivity of the slow -SH groups of the enzyme with 5, 5'-dithiobis (2-nitrobenzoic acid), suggesting that the environments of these groups are affected by the process. On the other hand, kinetic data show that the saturation of both classes of AMP binding sites affects the catalytic behavior and that their specific effects on the catalytic process are altered by the enzymatic transition dependent on the enzyme concentration. Thus, a strong inhibition is associated with the saturation of the weaker affinity AMP binding sites at 3mg/ml while the saturation of these weaker affinity binding sites at 1mg/ml produces an important activation of the enzyme.View full abstractDownload PDF (593K) -
Some Physico-Chemical and Enzymatic PropertiesShugo WATABE, Kanehisa HASHIMOTO1980 Volume 87 Issue 5 Pages 1491-1499
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA comparative study was performed on the myosins isolated from the white and dark muscles of the mackerel, Pneumatophorus japonicus japonicus.
The myosin from white muscle had three light chain subunits of molecular weights of 26, 500, 20, 000, and 17, 500. The myosin from dark muscle had only two light chains of molecular weights of 24, 000 and 19, 000. The former and the latter light chain patterns resemble those of the fast and slow muscle myosins of rabbit, respectively. Both mackerel myosins did not differ from each other significantly in sedimentation coefficient (about 6S) and amino acid composition.
The Ca2+-ATPase activity of dark muscle myosin was higher than that of white muscle myosin and the EDTA-ATPase activity of the former was lower than that of the latter irrespective of ionic strength at pH 7.0. The effects of Ca2+ and EDTA on both the kinetic constants, Km and Vmax, were similar for the two mackerel myosins. The pH optima of Ca2+-ATPase activity were observed at around 6.5 and 9.5 for white muscle myosin and only at around 6.0 for dark muscle myosin.
Actin, whether from white or dark muscle, enhanced the Mg2+-ATPase activity of each myosin significantly. This ATPase activity of synthetic actomyosin from the white muscle was about 6 times as great as that from the dark muscle counterpart.
The inactivation rate constant of ATPase activity of white and dark muscle myosins was 1.3×10-5 and 3.5×10-6 s-1 at 0°C, and 1.1×10-2 and 3.3×10-3s-1 at 30°C, respectively, indicating that the dark muscle myosin was 3.3-3.7 times more thermostable than the white muscle myosin.View full abstractDownload PDF (1320K) -
Shigeharu TAKIYA, Yoshiko TAKOH, Masaki IWABUCHI1980 Volume 87 Issue 5 Pages 1501-1509
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe template specificity of DNA-dependent RNA polymerases I and II (ribonucleoside 5'-triphosphate: RNA nucleotidyltransferase [EC 2. 7. 7. 6]) of Dictyostelium discoideum was investigated with several synthetic polynucleotides at three different stages of development. Both the enzymes exhibited several common characteristics for some templates, and distinctly different properties for other ones. Of single-stranded homopolymers, the strands of pyrimidine nucleotides were much transcribed in the order of poly (dC)>poly (dT). The doublestranded homopolymers, poly (dA)•poly (dT) and poly (dG)•poly (dC) were transcribed asymmetrically, the pyrimidine-containing strand being preferentially read. Transcription of double-stranded alternating copolymers, poly [d (A-T)]•poly [d (A-T)] and poly [d (G-C)]•poly [d (G-C)] occurred to some extent. Except for poly (rC), all of the single-stranded ribonucleotide homopolymers were extremely poor as templates. The polynucleotides containing thymidine were more efficient templates for polymerase I than polymerase II. The enzyme activities of the two polymerases were more or less variable with some polynucleotides among three stages of development, suggesting the possibility that D. discoideum RNA polymerases tend to change their template specificity during development.View full abstractDownload PDF (660K) -
Shuhei YASUDA, Yasuo KITAGAWA, Etsuro SUGIMOTO, Makoto KITO1980 Volume 87 Issue 5 Pages 1511-1517
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSErucic acid was not incorporated into phosphatidylcholine or phosphatidylethanolamine, but was into diphosphatidylglycerol and sphingomyelin in both the heart and liver of male Wistar rats fed erucic acid for 20 days.
A remarkable difference between the heart and liver was found in the molecular species composition of phosphatidylcholine. The 1-stearoyl//2-arachidonoyl species of phosphatidyl-choline in the heart was maintained at a higher level in rats fed the erucic acid diet than in rats fed a low fat diet. There was no difference in this molecular species content of cardiac and hepatic phosphatidylethanolamine between rats fed erucic acid and low fat diets.
The level of erucic acid incorporated into triacylglycerols and free fatty acids in the heart was higher than in the liver.View full abstractDownload PDF (445K) -
Yasunori KITAMOTO, Hiroshi MAEDA1980 Volume 87 Issue 5 Pages 1519-1530
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMany studies have been reported on the reaction of formaldehyde (FA) with amino acids or proteins, and FA is assumed to react with the α-amino group as well as some of the side chain groups. In most of these investigations a large excess of FA relative to amino acids or proteins was employed. In the present study, however, we carried out the reaction with a smaller excess of FA in order to clarify the reactivity, firstly with the α-amino groups, and secondly with specific side chain groups. No evidence for so-called Schiff base (-N=CH2) formation was obtained in the reaction with the α-amino groups, but the formation of an acid-labile N-hydroxymethyl compound as a major product was suggested by NMR and IR at ≥ pH 9.2. There was no indication, however, of the presence of such a product below pH 9.2, and the amount of N-hydroxymethyl product increased in parallel with the reaction pH. The higher the reaction pH (≥ pH 9.2), the greater the consumption of FA, up to 2 mol/mol Ala. In addition, the larger the excess of FA, the smaller the amount of free amino group remaining (at pH 9.7). In the assignment of IR spectra, discrete absorption bands of the α-carboxyl group of Ala were observed, which reflected ionization states of the α-amino group, and these were utilized for analysis of the reaction mechanism. Furthermore, among amino acids with side chain groups, His, Trp, and Arg showed high reactivity and Asn showed moderate reactivity. The products were relatively stable and were purified and subjected to instrumental analyses. Sixteen other amino acids including Tyr and Lys did not yield stable products. The products from Arg were unique because of the non-involvement of the amino group, and were reversibly converted to the original Arg upon acid hydrolysis. The products from His, Trp and Asn all involved amino or amide nitrogen forming cyclic ring structures with methylene derived from FA. The chemical structures of these products were determined on the basis of elemental analyses, MS and NMR.View full abstractDownload PDF (879K) -
Masafumi TSUJIMOTO, Keizo INOUE, Shoshichi NOJIMA1980 Volume 87 Issue 5 Pages 1531-1537
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCRP-induced agglutination of lipid suspensions prepared in the presence and absence of phosphatidylcholine was studied by a newly devised quantitative method.
CRP caused as much agglutination of suspensions composed of egg yolk phosphatidylcholine, cholesterol, and Span 60 as of those composed of cholesterol and Span 60, suggesting that phosphocholine residues of phosphatidylcholine are not important as binding sites for CRP. Agglutination of suspensions prepared without phosphatidylcholine was inhibited by phosphocholine, indicating that the inhibition by phosphocholine is non-competitive. Although phosphatidylcholine is not an essential component for agglutination of suspensions, it may modify the mode of interaction of CRP with its binding site on lipid suspensions, since the sensitivity of the agglutination to phosphocholine and the Ca2+ requirement were influenced by the presence of phosphatidylcholine in the suspensions.View full abstractDownload PDF (525K) -
Nobuhiro HARADA, Tsuneo OMURA1980 Volume 87 Issue 5 Pages 1539-1554
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The subcellular distribution of nitrobenzene reduction activity in rat liver cells indicated the existence of two different enzyme systems, one localized in microsomes and the other localized in cytosol. The activity in the cytosol was mainly attributable to xanthine oxidase, judging from its substrate specificity and the inhibition by allopurinol.
2. The participation of the microsomal electron transport system in nitrobenzene reduction was examined by using antibodies against four components of the system, NADPH-cytochrome c reductase (fpT), NADH-cytochrome b5 reductase (fpD), cytochrome b5, and cytochrome P-450. Both NADH- and NADPH-dependent nitrobenzene reduction activities were strongly inhibited by anti-fpT IG and also by anti-P450 IG, but not inhibited by anti-fpD IG or anti-b5 IG. The reduction of nitrosobenzene and phenylhydroxylamine, which are supposed to be the intermediates of nitrobenzene reduction, was also examined, and it was found that NADH- and NADPH-dependent reduction of both compounds were strongly inhibited by anti-fpT IG and anti-P450 IG, but not by anti-fpD IG or anti-b5 IG.
3. Reconstitution experiments using purified NADPH-cytochrome P-450 reductase and cytochrome P-450 were also carried out and it was confirmed that the reduction of nitrobenzene, nitrosobenzene, and phenylhydroxylamine to aniline could be effected by these two components.
4. Nitrobenzene reduction by microsomes exhibited a short initial time lag and was activated by the addition of purified NADPH-cytochrome c reductase, whereas nitrosobenzene and phenylhydroxylamine reductions did not show any initial time lag and were not activated by the reductase. These observations suggest that the reduction of nitrobenzene to an intermediate, possibly nitrosobenzene or phenylhydroxylamine, limits the rate of aniline formation, and such an initial step of nitrobenzene reduction can be catalyzed by NADPH-cytochrome c reductase alone. Cytochrome P-450 is essential at least in the final step of nitrobenzene reduction to aniline. This conclusion was further confirmed by determination of these intermediates in nitrobenzene reduction.View full abstractDownload PDF (1228K) -
I. Backone Chain Folding at 3 A ResolutionYoshiki MATSUURA, Masami KUSUNOKI, Wakako HARADA, Nobuo TANAKA, Yuichi ...1980 Volume 87 Issue 5 Pages 1555-1558
Published: May 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of Taka-amylase A was studied by an X-ray diffraction method at 3 A resolution. A total of 452 amino acid residues were found from the electron density map at the present stage. The four disulfide bonds and the branched carbohydrate were also located on the map. The difference electron density map of the maltotriose-soaked crystal showed that a maltose unit was bound in the active center cleft. The binding of iodine atoms to the enzyme was also studied.View full abstractDownload PDF (1729K)
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