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Volume 88 (1980)
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Volume 88, Issue 2
Displaying 1-40 of 40 articles from this issue
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Augustinus Rivai RINALDY1980 Volume 88 Issue 2 Pages 291-296
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis investigation deals both qualitatively and quantitatively with the changes of RNA content and synthesis during the culture growth cycle of Tetrahymena. Affinity chromatography with an oligo (dT) column was used to separate poly (A)+ RNA from total RNA. The rates of synthesis of poly (A)- and poly (A)+ RNA were determined in terms of the incorporation of [5-3H]uridine. During the log phase, the cellular RNA and protein contents decreased steadily, whereas during the resting stage, both were constant. The extents of decrease of both fractions of RNA were essentially the same (54.4% and 50.6% for total and poly (A)+ RNA, respectively). Therefore, the relative content of poly (A)+ RNA was constant from the beginning of the log to the resting stage (4.58%). The decrease in protein content, however, amounted to only 24.8%. Theoretically, a change in the age distribution during culture growth would cause a lower content of both fractions of RNA. The extents of the decrease in the rate of synthesis of both fractions were the same (75% and 79% for poly (A)- and poly (A)+ RNA, respectively). However, this reduction is so large that it cannot be solely the result of a shift of the age distribution of the cell population.View full abstractDownload PDF (437K) -
The Reactive Site Amino Acid Sequences of Silktree (Albizzia julibrissin) Seed Proteinase InhibitorsShoji ODANI, Teruo ONO, Tokuji IKENAKA1980 Volume 88 Issue 2 Pages 297-301
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo silktree (Albizzia julibrissin) seed proteinase inhibitors, A-II and B-II, which are homologous with soybean trypsin inhibitor (Kunitz), were cleaved with cyanogen bromide. Sequence analyses of the resulting fragments and comparison with the soybean inhibitor revealed the amino acid sequences around the reactive sites of both inhibitors. The primary reactive site of trypsin-chymotrypsin inhibitor A-II was identified as Arg66-Ile67 and that for chymotrypsin-elastase inhibitor B-II as Leu62-Met63. These results are the first reports of the reactive site sequences of homologs of soybean trypsin inhibitor (Kunitz).View full abstractDownload PDF (370K) -
Akihiko KAWAGUCHI, Kazumi ARAI, Yousuke SEYAMA, Tamio YAMAKAWA, Shigen ...1980 Volume 88 Issue 2 Pages 303-306
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pattern of fatty acids produced by the fatty acid synthetase complex of Brevibacterium ammoniagenes under several conditions was examined. The fatty acid synthetase obtained from B. ammoniagenes produced oleic acid as well as saturated fatty acids (palmitic and stearic acids). The relative proportions of palmitic to stearic acids varied over a wide range. Such alterations were dependent on the malonyl-CoA concentration and the ratio of acetyl-CoA to malonyl-CoA concentrations. At malonyl-CoA concentrations higher than 100 μm, stearic acid accounted for more than 90% of the saturated fatty acids and the pattern of fatty acid synthesized was independent on the ratio of acetyl-CoA to malonyl-CoA. At malonyl-CoA concentrations lower than 100 μm, raising the acetyl-CoA/malonyl-CoA ratio increased the percentage of palmitic acid. However, the proportion of oleic acid produced remained almost constant under all conditions tested.View full abstractDownload PDF (329K) -
Nobuo MIKI, Toshihiro SUGIYAMA, Toshi YAMANO1980 Volume 88 Issue 2 Pages 307-316
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA trypsin-solubilized form of cytochrome b5 has been coupled with Sepharose 6 B, and the immobilized cytochrome b5 was used as an affinity adsorbent for the purification of cytochrome P-450. Chromatography of the partially purified cytochrome P-450 on cytochrome b5-Sepharose 6 B and CM-Sephadex resulted in purification of cytochrome P-450 to a gel-electro-phoretically homogeneous state with an overall yield of 5% from microsomes of untreated rabbit livers. The purified cytochrome, designated as P-4503B1, had a specific content of 15 nmol per mg of protein. The minimum molecular weight of the cytochrome was estimated to be 52, 000 by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis, and neither the phenobarbital-inducible nor the 3-methylcholanthrene-inducible form of cytochrome P-450 co-migrated with this purified P-450B1.
Oxidized P-450B1, exhibited absorption maxima at 567, 533, and 417 nm, characteristic of a hemoprotein of low-spin type; the dithionite-reduced form showed absorption maxima at 545 and 413nm. The reduced, carbon monoxide-bound form had absorption peaks at 551 and 449nm. The electron paramagnetic resonance spectrum of the oxidized state of P-450B1 was that of a low-spin ferric hemoprotein with g values of 1.91, 2.24, and 2.40. The absolute spectrum of the reduced P-450B1-phenyl isocyanide complex exhibited absorption maxima at 549, 454, and 429 nm, and the absorbance ratio of A454 to A429 in the difference spectrum was 6.5 in 0.3M potassium phosphate, pH 7.4. The purified P-450B1, was active in the metabolism of benzphetamine, aminopyrine, and aniline with turnover numbers of 5.7, 6.4, and 2.7 nmol of product formed per min per nmol of cytochrome P-450, respectively.
The electrophoretic, spectral, and catalytic properties together with the uniquely high affinity of P-4503, for cytochrome b5 indicate that the purified P-450B1, is a new form of cytochrome P-450, distinct from those previously reported.View full abstractDownload PDF (759K) -
Tatsuo NAKAYAMA, Yumiko KUROGI, Maki IRIKURA, Hisayuki MATSUO, Aiko SA ...1980 Volume 88 Issue 2 Pages 317-326
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lysozyme-detergent method was developed for the fractionation of sporulating cells of B. subtilis 168 wild type into mother cell and forespore fractions. The method is very mild and is reproducible with optimum concentrations of Brij-58, deoxycholic acid and sucrose. The results were confirmed by application of the method to temperature sensitive mutants, Ts-1 and Ts-3.
The amounts of proteins, and the activities of protease, alkaline phosphatase and glucose dehydrogenase were about 55, 56, 91, and 40%, respectively, in the mother cell fraction, and about 45, 44, 9, and 60%, respectively, in the forespore fraction, taking the totals for the combined fractions as 100%.
Slab gel electrophoretic patterns indicated that many species of proteins with different molecular weights were present in the two fractions.
Pulse-labeling with [3H]UTP was carried out in vivo at stage III, and 35.2 and 64.8% of the [3H]UMP incorporated into RNAs were distributed in the mother cell and forespore fractions, respectively. The results indicate that more RNA synthesis occurs in the forespores than in the mother cells of sporulating cells.View full abstractDownload PDF (1876K) -
Makoto NAKAO, Yoshimi FUJII, Yukichi HARA, Takeo NOMURA, Toshiko NAKAO ...1980 Volume 88 Issue 2 Pages 327-335
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral techniques were devised to estimate the phosphorylation capacity of spectrin (band 2 according to Fairbank's numbering) using intact erythrocytes. 32P incorporation from inorganic phosphate into erythrocytes was accelerated by eliminating chloride ions from the incubation media. The simultaneous addition of adenine and inosine accelerated the 32P incor-poration into ATP and effectively maintained the level of ATP in the cell as well as the specific radioactivity of ATP labile phosphate. Incubation was carried out using citrate medium or calcium-sucrose medium with 32P, adenine, and inosine. After incubation for 2, 6, or 20 h, the radioactivity of labile P of ATP in the cell was assayed by acid extraction, absorption on plastic-coated charcoal, elution from the charcoal, removal of the solvent, hydrolysis in 1 N HCl and extraction with organic solvent as a phosphate-molybdate complex. Membrane proteins in the hemolysate precipitate were subjected to slab sodium dodecyl sulfate gel elec-trophoresis according to Laemmli and Fairbanks. After staining with Coomassie Blue, the gels were dried on transparent sheets. Radioactivity was counted through a specifically devised transparent slit, and after densitometry a radioautogram was taken on X-ray film. The amount of phosphate incorporated into band 2 was calculated from the cpm/mol value for band 2 (not band 1+2) and the specific radioactivity of ATP labile phosphate.
The amount of ATP in the cell and the specific radioactivity of ATP labile P remained nearly constant for 6 and 20 h, respectively. The maximum 32P incorporation into band 2 of normal intact cells was 5.03±1.94 mol/mol of band 2 (citrate medium) (n=7) and 8.25±1.73 (Ca-sucrose medium) (n=4). These are larger than the values previously reported. The value for hereditary spherocytotic cells was over 22.6+5.75 (control 7.4±2.86) in 3 cases in the presence of calcium in the medium and 6.65±1.42 (control 3.9±1.07) in the citrate medium (4 cases). These results are quite different from the observations of other workers. 32P incorporation into cell membranes, which was determined by hemolysis, was below 1, which is similar to values given in other reports.
32P incorporation into other membrane proteins was also observed by taking radioauto-grams. All radioactive bands other than 2, 21, 23, 25, 29, and hemoglobin were different from the Coomassie Blue-stained bands. The incorporation was greatest at band 2. The bands of 21, 23, 25, and 29 also showed high specific activity, but the amounts of these proteins were not great. The radioactivities of all these bands seemed to be a little higher in spherocytic cells.View full abstractDownload PDF (709K) -
Yuji OHYAMA, Hiroichi FUKAMI, Takahisa OHTA1980 Volume 88 Issue 2 Pages 337-348
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly (A) polymerase [EC 2. 7. 7. 19] was highly purified from beef liver nuclei by the use of column chromatographies on heparin-Sepharose 4 B and Blue Dextran-Sepharose 4 B. The purified enzyme showed one major protein band of the molecular weight of 57, 000 in SDS polyacrylamide gel electrophoresis, which agreed with the molecular weight estimated from glycerol gradient centrifugation. The enzyme required the presence of Mn2+ for its activity but was almost completely inactive with Mg2+. It incorporated specifically ATP into polynucleotide as a sole substrate. The enzyme activity depended entirely on the addition of exogenous polynucleotide primer. It showed certain selectivity for the primers. The most effective among the tested polynucleotides was a short poly (A), for which the Km of the enzyme was shown to be 7μM. Poly (G, U) and short poly (U) also primed the reaction, but tRNA, phage RNA, poly (G), and poly (C) were inactive. Based on observed specificity for the primer, the role of this enzyme in the cell nuclei was discussed. Digestion of the reaction product of this enzyme by two specific exonucleases, snake venom and spleen phospho-diesterases, suggested that this enzyme catalyzed the covalent bonding of the substrate to the 3' terminus of the primer as in the manner expected for in vivo polyadenylation.View full abstractDownload PDF (864K) -
Michio IMAWARI, Yasuo AKANUMA, Yasutoshi MUTO, Hiroshige ITAKURA, Kino ...1980 Volume 88 Issue 2 Pages 349-360
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo immunologically similar, probably identical, binding proteins for vitamin D and its metabolites (DBP1 and DBP2) were isolated separately from rat serum after approximately 180-fold purification by novel procedures using Blue Sepharose CL-6B chromatography. The freshly purified DBP1 and DBP2 each showed a single band of protein on polyacrylamide gel electrophoresis, and had α-mobility, although DBP1 moved slightly faster than DBP2. DBP1 and DBP2 had the same molecular weight, which was estimated as approximately 54, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric points of DBP1 and DBP2 were estimated as 4.9 and 5.0, respectively, from the results of isoelectric focusing experiments. DBP1 and DBP2 both appeared to have one binding site for 25-hydroxyvitamin D3 per molecule of protein, with apparently similar association constants at 4°C of 5-7×109M-1 The amino acid compositions of DBP1 and DBP2 were also determined and compared. A monospecific antiserum against rat DBP2 was prepared in a rabbit and was used for immunological studies of rat DBP. On double immunodiffusion, anti-DBP2 antiserum produced precipitin lines of complete reaction-of-identity against the purified DBP1, the purified DBP2, and rat whole serum. There was no immunological cross-reactivity between rat DBP and sera from man, dog, and rabbit, but mouse serum showed a pattern of partial identity with rat DBP. When rat serum samples were analyzed by immunoelectrophoresis using anti-DBP antiserum, three patterns of precipitin line were observed: a pattern showing the existence of only DBP1, designated as DBP 1-1; a pattern showing the existence of only DBP2, designated as DBP 2-2; and a pattern showing the existence of both DBP1 and DBP2, designated as DBP 2-1. Using single radial immunodiffusion assay for rat serum DBP, the mean (±S. D.) serum DBP concentrations were found to be 461±59 μg/ml in adult male rats and 328±16 μg/ml in adult female rats, and the difference was significant (p<0.001). In molar terms, DBPs are present in normal rat serum in large excess relative to vitamin D and its metabolites, and most of the serum DBP, therefore, circulates as apo-DBP, not containing abound molecule of vitamin D or of its metabolites. The immunoprecipitation studies of DBP in rat serum showed that DBPs were common main transport proteins for naturally occurring vitamin D and its metabolites, and that DBP played some, but not a principal, role in the transport of synthetic 1α-hydroxyvitamin D3.View full abstractDownload PDF (2654K) -
Determination of Cleavable Points and Their CharacterizationYoh OKAMOTO, Mami OKAMOTO, Takamitsu SEKINE1980 Volume 88 Issue 2 Pages 361-371
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe locations of ATP-induced structural change in gizzard myosin were examined by analyzing the changes in the chymotryptic fragmentation pattern. From the time course of fragmentation, and by fractionation of the produced fragments and detection of the masked amino terminal fragment, the original positions of the six different fragments in the parent myosin molecule were assigned. A reconstituted model of myosin based on the above assignment showed the presence of three cleavable sites in the heavy chain of myosin. ATP accelerated the cleavage at site 1, 5 K daltons from the masked amino terminus, while it inhibited cleavage at site 2, 71 K daltons from the N terminus. These opposite effects of ATP on sites 1 and 2 were remarkable, but ATP had no significant effect on cleavage at site 3, 100 K daltons from the carboxyl terminus. These results indicate that two distant regions in the myosin head, 66 K daltons apart in the primary sequence, were exposed or buried with the progress of the ATPase reaction.
In addition, prolonged chymotryptic digestion failed to produce any subfragment-1, irrespective of the presence or absence of divalent cations in the digestion medium, but produced a protease-resistant and relatively long (100 K daltons2) light meromyosin. This suggests a distinctive feature of the neck and hinge regions in gizzard myosin.View full abstractDownload PDF (3061K) -
Bernard Y. HIRAOKA, Katsuhiko FUKASAWA, Kayoko M. FUKASAWA, Minoru HAR ...1980 Volume 88 Issue 2 Pages 373-377
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA unique phosphoprotein, which contains an uncommon amino acid, α-amino adipic acid (α-AAA), was isolated from unerupted bovine teeth by extensive EDTA demineralization. The α-AAA in the protein hydrolysates was identified based on the elution time on an amino acid analyzer using the sodium and lithium citrate buffer in a dual column system, and its identity was confirmed by comparison of its DNS derivative with that of authentic α-AAA. This result suggests that the lysine residues in the phosphoprotein may be deaminated by an amine oxidase into allysine and further oxidized to α-AAA.View full abstractDownload PDF (978K) -
Masaaki TAGUCHI, Katsura IZUI, Hirohiko KATSUKI1980 Volume 88 Issue 2 Pages 379-387
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen Escherichia coli strain CP78 (rel+) was starved for isoleucine by the addition of valine, the amount of glucose in polymeric form in the cells increased markedly compared to that of the control cells. In contrast, this phenomenon was not seen in strain CP79 (rel-). The increase in CP78 was shown to be due to the increase of glycogen. These results indicate that glycogen synthesis was augmented under stringent control. This was confirmed using other isogenic pairs of rel+ and rel- strains starved for other amino acids.
When the cultivation temperature of strains 10B601 (rel+) and 10B602 (rel-) possessing temperature-sensitive valyl-tRNA synthetase was shifted from 30°C to 40°C, no difference was observed in the response of glycogen synthesis between the two strains. These results indicate that protein synthesis was necessary for the augmentation of glycogen synthesis and that guanosine 5'-diphosphate 3'-diphosphate did not exert its effect through stimulation of the activity of pre-existing enzyme (s) involved in glycogen synthesis. These conclusions were supported by the results of experiments using chloramphenicol and rifampicin.
The rates of glucose utilization of CP78 and CP79 were decreased to nearly the same extent by valine addition. This suggests that the regulation site of glycogen synthesis under stringent control resides in a step after the transport of glucose by the phosphotransferase system.View full abstractDownload PDF (734K) -
Yu HOSOKAWA, Kenji YAMAGUCHI, Noriyuki KOHASHI, Yoshihiko KORI, Osamu ...1980 Volume 88 Issue 2 Pages 389-394
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCysteine dioxygenase activity was not detected in the liver of either fetal or 2-day-old rat. The enzyme activity of neonatal rat liver gradually increased between the 4 th and 12 th postnatal days and then sharply increased to reach the adult level by the 28 th postnatal day. No cysteine dioxygenase activity was observed in the fetal liver at the 18 th day of gestation from a dam injected with hydrocortisone. Hydrocortisone-mediated induction was first observed in the livers of 4-day-old rats. The duration of hydrocortisone-mediated induction was much longer in the livers of 4- to 12-day-old rats than in those of adult rats. The enzyme half-life in livers of 10- and 20-day-old rats was 6.4 and 3.7 h, respectively. The half-life of the enzyme in 20-day-old rat liver was increased to 6.2 h by L-cysteine injection.View full abstractDownload PDF (425K) -
Hiroshi KIMURA, Hisae MURATA, Heima UEMATSU1980 Volume 88 Issue 2 Pages 395-401
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Native human hemoglobin A was digested with proteases after treatment with reduced or oxidized glutathione, cysteine, or cystine.
2. The treatment of hemoglobin with a 40-fold molar excess of reduced glutathione or cysteine at 37°C for 4 h enhanced its susceptibility to trypsin 4 or 6 times, respectively. However, oxidized glutathione and cystine had little or no effect on its susceptibility to the protease.
3. The enhanced susceptibility was not due to conformational change in hemoglobin, such as dissociation to dimers or monomers, or to unfolding of α-helical structure, but was due to dissociation of heme from the globin moiety, judging from measurements of the absorption spectrum and circular dichroism spectrum, and gel-filtration analysis.View full abstractDownload PDF (508K) -
Resonance Raman Spectra of Lumiflavin and 8-Substituted RiboflavinYasuzo NISHINA, Kiyoshi SHIGA, Kihachiro HORIIKE, Hiromasa TOJO, Sabu ...1980 Volume 88 Issue 2 Pages 403-409
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe resonance Raman (RR) spectra of 8-halogenated-riboflavin, 8-demethyl-riboflavin (8-H-RF), 8-amino-riboflavin (8-NH2-RF), 8-methoxy-riboflavin (8-OCH3-RF), lumiflavin, and 3-methyl-lumiflavin were observed. The Raman lines with the highest frequency are at 1624,
1620, and 1615 cm-1 for 8-chloro-riboflavin, 8-bromo-riboflavin, and 8-iodo-riboflavin, respectively. This systematic shift confirms that the 1631 cm-1 line of riboflavin is derived from the benzene part of isoalloxazine. Substitution at the 8-position by an amino or methoxy group, which has a large influence on the electronic structure of isoalloxazine, changes the RR spectrum markedly in comparison with that of 8-halogenated riboflavin. The 1583 cm-1 line of riboflavin, which involves the vibrational displacement of N (5) and C (4a) atoms of isoalloxazine, is shifted to the low frequency side by substitution at the 8-position with an amino or methoxy group. The corresponding line of 8-H-RF, on the contrary, shifts to the high frequency side. The RR spectrum of lumiflavin is very different from that of riboflavin in the range from 1200 to 1300 cm-1. Although the π-electronic structure is little affected by the substitution at the 10-position, the Raman spectrum of lumiflavin in this region is very sensitive.View full abstractDownload PDF (434K) -
Yasuzo NISHINA, Kiyoshi SHIGA, Kihachiro HORIIKE, Hiromasa TOJO, Sabu ...1980 Volume 88 Issue 2 Pages 411-416
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe resonance Raman (RR) spectra of semiquinones of complexes of riboflavin or 8-methoxy-riboflavin (8-OCH3-RF) with riboflavin binding protein (RBP) were observed. The RR spectrum of neutral semiquinone of riboflavin-RBP complex in H2O solution has an intense line at 1617cm-1, not observed for oxidized riboflavin bound to RBP. The line at 1617cm-1 does not shift in D2O solution. The absorption spectrum of semiquinone of 8-OCH3-RF bound to RBP has maxima at 586, 396, and 344 nm, and the RR spectrum doublet lines at 1623 and 1615 cm-1. In D2O solution, the 1623cm-1 line does not shift, but the 1615cm-1 line shifts to 1604cm-1 The line around 1620cm-1 for the flavin semiquinone will be useful in the determination of the redox state of flavin.View full abstractDownload PDF (344K) -
Hidenari TAKAHARA, Hitoshi NAKAYAMA, Hyogo SINOHARA1980 Volume 88 Issue 2 Pages 417-424
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-1-Antitrypsin was purified from rat plasma using affinity chromatography on Cibacron Blue-agarose, ion-exchange chromatography on DE-52 (pH 8.5 and 6.0) and gel filtration on Sephadex G-150. The final preparation was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, by immunoelectrophoresis and by analytical ultracentrifugation. It was composed of a single polypeptide chain whose molecular weight was estimated to be about 50, 000 by sedimentation equilibrium analysis. Rat α-1-antitrypsin was shown to be a glycoprotein containing 3.3% glucosamine, 1.7% mannose, 1.0% galactose, and 4.2% sialic acid.
The interaction of rat α-1-antitrypsin with bovine β-trypsin was studied by protease activity assays and by gel electrophoresis in sodium dodecyl sulfate. Rat α-1-antitrypsin inhibited bovine β-trypsin by forming a stable equimolar complex concomitant with the release of a peptide with a molecular weight of approx. 8, 000.View full abstractDownload PDF (2791K) -
Michiteru YOSHIDA, Yoshihiro OHTAKI, Takashi FUJITA, Kensuke SHIMURA1980 Volume 88 Issue 2 Pages 425-436
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. To establish a practical method to prepare nonhistone chromosomal proteins (NHP's) in substantial quantity, a sequential group fractionation procedure for NHP's from pig thymus chromatin has been developed by a combination of methods based on the intrinsic properties, such as binding force in chromatin, molecular weight, and acidity and/or basicity of the protein components.
2. The loosely bound NHP's in chromatin have been grouped into 11 subfractions, and tightly bound ones into 6 subfractions, 2 of which were histones. The loosely bound NHP's were chiefly acidic to neutral in contrast with the tightly bound components, which were basic. In addition, the NHP's of high molecular weight were in general acidic to neutral, while the lower molecular weight components were mainly basic.
3. The present method is applicable to the preparation of representative fractions of NHP on the scale of 50-400 mg from 1kg of pig thymus.View full abstractDownload PDF (1961K) -
Toshiaki ODA, Tsuneo OMURA1980 Volume 88 Issue 2 Pages 437-442
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFree ribosomes and membrane-bound ribosomes were prepared from rat livers, and the contributions of these two types of ribosomes to the synthesis of aldolase B were studied by the immunoprecipitation of [3H] puromycin-labeled nascent peptides with a rabbit antibody to this enzyme. Although rat liver aldolase was recovered in both cytosolic and microsomal fractions by the fractionation of liver homogenate, the microsomal aldolase was immunologically identical with its cytosolic counterpart as confirmed by Ouchterlony immunodiffusion test. We examined the nascent peptide fractions prepared from free and bound ribosomes, and found that the nascent peptides of aldolase were mainly localized in free ribosomes. About 0.5% of the total nascent peptides of free, ribosomes and 0.08% of those of bound ribosomes was aldolase. The site of synthesis of serum albumin was also examined as a reference standard by the immunoprecipitation of labeled nascent peptides, and the nascent peptides of this secretory protein were mainly associated with bound ribosomes, as reported by other workers. These observations confirm that aldolase B is mainly synthesized by free ribosomes in rat liver cells.View full abstractDownload PDF (1828K) -
Katsutoshi ISHIMARU, Hiroshi KIHARA, Motonori OHNO1980 Volume 88 Issue 2 Pages 443-451
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhospholipase A was purified from venom of Trimeresurus fiavoviridis (Habu snake) via three steps consisting of Sephadex G-100, CM-cellulose, and DEAE-cellulose column chromatographies. The apparent molecular weights determined by gel filtration on Sephadex G-75 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 28, 000 and 14, 000, respectively, suggesting that the enzyme is composed of two identical subunits. The isoelectric point was 7.9. The enzyme is characterized by high contents of aspartic acid, glycine, tyrosine, and lysine and contains one residue each of histidine and methionine, three or four tryptophans, and eight disulfide bonds per subunit. The enzyme was inactivated by reaction with p-bromophenacyl bromide following pseudo first order kinetics. The loss of activity was accompanied by loss of histidine, indicating that a single histidine residue is essential for activity. Oxidation with N-bromosuccinimide decreased the enzymic activity. Tryptophan residues appear to play some role in catalysis.View full abstractDownload PDF (1105K) -
Takao YAGI, Yasuo MUKOHATA1980 Volume 88 Issue 2 Pages 453-461
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of the presence of digitonin in the reaction mixture on photophosphorylation, light-induced H+ uptake, and the size of isolated spinach chloroplasts were studied. Digitonin inactivated photosystem II and dissipated light-induced pH increase without affecting the photophosphorylation driven by photosystem I. Digitonin increased the concentration of NH4CI required for uncoupling. This effect of digitonin was diminished by valinomycin which barely affects phosphorylation by itself. Although such properties have all been observed with digitonin subchloroplast particles, digitonin at the concentration required for acquiring such properties had little effect on the size of chloroplasts. These results suggest that the characteristic effects of digitonin subchloroplast particles are caused by thylakoid membranes penetrated by digitonin but not by the size of membrane vesicles.
Both in chloroplasts with digitonin in the reaction mixture and in digitonin subchloroplast particles, N, N'-dicyclohexylcarbodiimide, purine nucleoside di- and tri-phosphates, and organic bases with low pKa values (pyridine and aniline) made the light-induced pH increase detectable, and this was lost upon the addition of proton ionophores. These results suggest that the lack of the light-induced pH increase by chloroplasts with digitonin in the reaction mixture and by digitonin subchloroplast particles is caused both by a membrane leaky to protons and by the loss of internal buffering capacity of thylakoids. Furthermore, the phosphorylation of chloroplasts with digitonin in the reaction mixture and of digitonin subchloroplast particles is considered to be driven by the proton motive force as is that of chloroplasts without digitonin.View full abstractDownload PDF (1173K) -
Yukuo YOSHIDA, Eisuke FURUYA, Kunio TAGAWA1980 Volume 88 Issue 2 Pages 463-468
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and sensitive colorimetric method for the quantitative determination of phospholipids in various kinds of biological materials is described. The method, a modification of a previous method using tetrathiocyanatocobaltate, is based on the formation of hydrophobic complexes between dithiocyanatoiron reagent and phospholipids. Under the standard conditions all species of neutral phospholipids, including lysophosphatidylcholine and sphingomyelin, form stable hydrophobic complexes with the iron compound. Although species of acidic phospholipids also have slight affinity for the iron compound, they do not disturb the assay appreciably, because their contents in biological materials are low. Under special conditions, the iron compound reacts only with phosphatidylcholine, and therefore it may also be used for specific determination of this phospholipid.View full abstractDownload PDF (421K) -
Yoshio ARAKI, Shozo OBA, Satoko ARAKI, Eiji ITO1980 Volume 88 Issue 2 Pages 469-479
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme which catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine residues in cell wall peptidoglycan was found in the supernatant and 20, 000×g pellet fractions of Bacillus cereus. Autolysis of the latter fraction resulted in solubilization and activation of the deacetylase. Among various bacteria, strains of B. cereus which contain high proportions of N-unsubstituted glucosamine residues in their cell wall peptidoglycan components are particularly rich in the deacetylase.
The peptidoglycan deacetylase is distinguishable from N-acetylglucosamine-6-phosphate deacetylase [EC 3. 5. 1. 25] on the basis of their cellular distribution and chromatographic behavior. The rate of reaction of the deacetylase with (N-acetylglucosaminyl-N-acetylmuramic acid)3 [abbreviated as (GlcNAc-MurNAc)3] is less than 1/100 of that with peptidoglycan, while the enzyme is inactive towards (GlcNAc-MurNAc)2, GlcNAc-MurNAc, and monomeric N-acetylglucosamine derivatives. The enzyme also deacetylates partially O-hydroxyethylated chitin. The concentrations of peptidoglycan and partially O-hydroxyethylated chitin required for half-maximum activities were found to be 0.29 and 6.9mg per ml (or 0.17 and 20mM with respect to N-acetylglucosamine residues), respectively. The occurrence of this enzyme accounts for the formation of cell wall peptidoglycan N-unsubstituted at the glucosamine residues.View full abstractDownload PDF (814K) -
Masato OKADA, Seiichi KAWASHIMA, Kazutomo IMAHORI1980 Volume 88 Issue 2 Pages 481-488
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Putrescine oxidase [EC 1. 4. 3. 10] was partially inactivated by reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), rapidly. The activity of the modified enzyme toward putrescine was 5.6% of that of the native enzyme. Moreover, the optimal pH and Km value of the modified enzyme for putrescine changed from 9.0 to 10.5 and from 0.23 to 3.4mM, respectively. This modified enzyme showed activity toward monoamines such as n-butylamine, n-hexylamine, and n-octylamine, which are not substrates of the native enzyme.
2. Heavy metal ions inactivated putrescine oxidase completely. Hg2+ had the stronger ability to inactivate it. This inactivation was due to the dissociation of FAD from the enzyme molecule. The FAD-free enzyme had no ability to reassociate FAD, in contrast to the apoenzyme. The enzyme treated with Hg2+ showed characteristic behavior on SDS-polyacrylamide gel electrophoresis.
3. From the results of chemical modification it was deduced that a carboxyl group plays an important role in binding the substrate.View full abstractDownload PDF (1288K) -
I. Resolution, Purification, and Molecular PropertiesYoshio IMAI, Chikako HASHIMOTO-YUTSUDO, Haruhiko SATAKE, Agnés ...1980 Volume 88 Issue 2 Pages 489-503
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method is described for the separation and purification of different forms of cytochrome P-450 from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-pretreated rabbits. It consists of solubilization of microsomes with cholate, followed by successive chromatography on ω-aminooctyl Sepharose 4 B, hydroxylapatite and CM-Sephadex C-50 columns. Separation of different forms of cytochrome P-450 is achieved in the aminooctyl Sepharose and hydroxylapatite chromatography steps. This method permits the separation of three forms of cytochrome P-450, i.e. “P-4501, ” “P-4502, ” and “P-4481, ” from PB-induced microsomes; P-4501, the main cytochrome P-450 component in these microsomes, and P-4481 can each be obtained in a gel-electrophoretically homogeneous state, whereas P-4502 can be obtained in a partially purified state. Application of the same method to MC-induced microsomes led to the purification of P-4481, the main component in these microsomes, to homogeneity and to partial purification of a fourth form, i.e. “P-4503.” P-4481 from MC-induced microsomes seems to be identical with P-4481 from PB-induced microsomes in monomeric molecular weight (54, 000), amino acid composition and chromatographic behavior. However, P-4481 from MC-induced microsomes, but not P-4481 from PB-induced microsomes, contains 0.18 to 0.88 mol of tightly bound MC per mol of protein. P-4501 has a molecular weight of 49, 000 and its amino acid composition is clearly different from that of P-4481. Although P-4502 is similar to P-4501 in molecular weight, they differ from each other in chromatographic behavior. P-4503 seems to be different from P-4501 in molecular weight, though they are similar to each other in chromatographic behavior. All the cytochrome P-450 preparations can be freed from the detergents used in the purification procedure by CM-Sephadex C-50 chromatography. Detergent-free P-4501, P-4502, and P-4481 exist in aqueous solution as oligomeric aggregates.View full abstractDownload PDF (1161K) -
II. Spectral PropertiesChikako HASHIMOTO-YUTSUDO, Yoshio IMAI, Ryo SATO1980 Volume 88 Issue 2 Pages 505-516
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe spectral properties of the multiple forms of cytochrome P-450 purified or partially purified from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rabbits have been studied. Both optical absorption and EPR studies have shown that the oxidized forms of P-4501, P-4502 (from PB-treated animals), and P-4503 (from MC-treated animals) are in the low spin state, having a Soret absorption peak at 417-418 nm. Oxidized P-4481 (from both PB- and MC-treated animals), on the other hand, shows a Soret peak at 393 nm and a weak band at 646 nm. This and EPR evidence indicate that P-4481 contains heme which is predominantly in the high spin state, though EPR studies at low temperature indicate the presence of a small amount of low spin ferric heme. The presence of tightly bound MC in P-4481 purified from MC-treated animals is reflected by characteristic absorption peaks in the ultraviolet region, but this does not affect the absorption spectra in the Soret and visible regions. Emulgen 913, a nonionic detergent, causes the conversion of oxidized P-4481 from the high to the low spin state, as evidenced by optical absorption and EPR results; bound MC inhibits this conversion in a noncompetitive way. Binding of ethyl isocyanide to reduced P-4501 and P-4481 results in the appearance of two Soret peaks in the 430 and 455 nm regions, the relative intensities of which are dependent on pH. At any pH the 455 nm peak of P-4481 is always higher than that of P-4501. Benzphetamine and aniline, added to oxidized P-4501, cause Type I and Type II spectral changes, respectively, but the magnitudes of the changes are small in both cases. The Soret peak of oxidized P-4481 at 393 nm is completely shifted to 420 nm on addition of aniline, resulting in a reverse Type I spectral change; acetanilide causes the conversion of the Soret peak to the low spin state to only a slight extent. The conversions caused by aniline and acetanilide are both inhibited by the presence of tightly bound MC. On the basis of these and other observations, the spin states of these P-450's are discussed.View full abstractDownload PDF (897K) -
Restriction of the Chain LengthKiyokazu MORIOKA, Kiyoko TANAKA, Tetsuo ONO1980 Volume 88 Issue 2 Pages 517-524
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Poly(ADP-ribose) synthesized in nuclei isolated from Friend erythroleukemic cells was characterized. Although the activity of poly(ADP-ribose) synthesis of cells induced to differentiate was suppressed to about one-third of that of uninduced cells, the chain lengths were identical, and were clearly restricted within a narrow band in polyacrylamide gels, moving at about half the speed of bromphenol blue.
2. The restriction of chain length was also observed in rat liver nuclei, though some poly(ADP-ribose) chains could be degraded into smaller pieces during incubation. Treatment of nuclei with a high concentration of detergent and sonication before incubation caused aberrant synthesis of poly(ADP-ribose) chains which were not restricted in size.
3. In contrast, the average chain length of the 3H-labeled portion of poly(ADP-ribose) synthesized during incubation of nuclei with 3H-NAD+ was found to be reduced to 50% of that in the induced cells.
4. Under our reaction conditions, about 95 % of newly synthesized poly(ADP-ribose) was linked to non-histone nuclear proteins. The total content of the non-histones was reduced to about 50% by treatment with inducers.
5. In view of the above findings, it is suggested that the decrease of chain initiation frequency caused by the partial loss of available acceptor sites may be correlated with the suppression of poly(ADP-ribose) synthesis in induced cells.View full abstractDownload PDF (611K) -
Jun-ichi AZUMA, Junji MORITA, Tohru KOMANO1980 Volume 88 Issue 2 Pages 525-532
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ØX 174-DNA membrane complex was isolated from Escherichia coli infected with ØX 174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The ØX 174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having buoyant densities of 1.195 and 1.150g/ml, respectively. Immediately after infection with ØX 147, replicating DNA was pulselabeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The ØX 174 DNA-membrane complex contained a specific membrane-bound protein having a molecular weight of 80, 000 which is accumulated in the host DNA-membrane complex. These results suggest that when ØX 174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.View full abstractDownload PDF (1217K) -
Mieko IWAI, Yuji SHIMADA, Yoshio TSUJISAKA1980 Volume 88 Issue 2 Pages 533-538
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRhizopus delemar (ATCC 34612) lipase was modified by phospholipid (PL)-treatment so as to enhance its activity on lipoprotein. In order to detect change in lipase conformation in the modified state, a preliminary experiment was performed to remove PL from the PL-treated lipase solution which included PL nonessential to enhancement of lipoprotein lipase (LPL) activity. It was found that treatment with a mixture of isopropyl ether: n-butanol (3:1) was suitable for this purpose because of the stability of the enzyme.
Changes in isoelectric point and α-helical content of lipase caused by PL-treatment were studied by means of isoelectric focusing and circular dichroism spectrum. The isoelectric point of lipase was found to shift to the acidic side on its binding with phosphatidylcholine (PC) or cardiolipin (CL). The circular dichroism spectra of the original lipase and PL-treated lipases indicated that the α-helical content of lipase decreased on its binding with PL.
In CL-bound lipase, which was more greatly enhanced as to LPL activity than was PC-bound lipase, α-helical content was decreased to a larger extent than that of PC-treated lipase.View full abstractDownload PDF (443K) -
Yasuo SUZUKI, Toshiro MOCHIZUKI, Makoto MATSUMOTO1980 Volume 88 Issue 2 Pages 539-546
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChange of lipid metabolism in FL cells derived from human amniotic membrane during fusion from within induced by measles virus was studied. The following results were obtained.
(1) Incorporation of [32P]orthophosphate and [methyl-14C]choline into the lipid fraction of infected cells was higher than that of control cells. In this case, no detectable alteration of labeled phospholipid composition was observed. (2) Radioactivity of the lipids in subcellular fractions of infected-cells labeled with [1-14C]oleic acid for a short term also increased. (3) It was confirmed that specific enzyme activities in phosphatidylcholine synthesis, i.e. ATP: choline phosphotransferase [EC 2. 7. 1. 32], CTP: phosphocholine cytidyltransferase [EC 2. 7. 7. 15], and CDPcholine: 1, 2-diacylglycerol cholinephosphotransferase [EC 2. 7. 8. 2] in each subcellular fraction of the infected-cells were higher than those of control cells. (4) AcylCoA: 1-acyl-GPC acyltransferase activity in microsomal fraction of the infected-cells was enhanced. The above results indicate that there is stimulation of cellular phospholipid synthesis in de novo pathway and acylation of lysophospholipids in the infected-cells.
A marked increase in radioactivity-incorporation (10.3 fold) of [1-14C]oleic acid into triacylglycerol in the cytosol of the infected cells was also observed.View full abstractDownload PDF (594K) -
Takayoshi IIO, Hiroshi KONDO1980 Volume 88 Issue 2 Pages 547-556
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComparison of the kinetic properties of troponin-C and dansylaziridine-labeled troponin-C revealed that the calcium ion binding and removal reactions with the low affinity Ca2+-binding sites (sites I and II) and the resultant local conformational change are rapid processes, and that the calcium ion binding and removal reactions with the high affinity Ca2+ binding sites (sites III and IV) are also rapid, whereas the resultant local conformational change is a slow process. It is also shown in this work that the latter processes are independent of the former processes.View full abstractDownload PDF (733K) -
Tadashi YOSHIDA, Masato NOGUCHI, Goro KIKUCHI1980 Volume 88 Issue 2 Pages 557-563
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new intermediate of heme degradation was found in the heme oxygenase reaction with the heme•heme oxygenase complex and the NADPH-cytochrome c reductase system. The intermediate showed an absorption peak at 688 nm and its carbon monoxide complex gave a peak at 638 nm. The 688 nm substance seemed to have an extremely high affinity for carbon monoxide, and when the reaction was performed under a gas mixture of carbon monoxide and air, the reaction stopped almost completely at the level of the 638 nm substance. The same intermediate also appeared when the heme•heme oxygenase complex was incubated with ascorbic acid. The carbon monoxide complex of the 688 nm substance was easily and quantitatively converted to biliverdin-iron chelate when exposed to air. The heme moiety of the 688 nm substance was extracted in chloroform and compared with protoheme and verdohemochrome by thin layer chromatography. The sample migrated faster than either verdoheme or protoheme. The pyridine complex of the heme moiety of the 688 nm substance was not converted to pyridine verdohemochrome when incubated in air in the presence or absence of ascorbic acid, indicating that the 688 nm substance is not involved in the intermediary step of heme degradation in the coupled oxidation of pyridine-hemin. In other words, the sequence of heme degradation catalyzed by the heme oxygenase system may differ in some respects from that in the coupled oxidation of pyridine-hemin. In addition, biliverdiniron chelate failed to afford pyridine verdohemochrome on the addition of pyridine. The chemical nature of the new intermediate remains to be elucidated.View full abstractDownload PDF (513K) -
Priska SPIRI-NAKAGAWA, Ruiko OIWA, Yoshitake TANAKA, Haruo TANAKA, Sat ...1980 Volume 88 Issue 2 Pages 565-570
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAzureomycin B (10 μg/ml), a new antibiotic from Pseudonocardia azurea nov. sp., caused the accumulation of lipid intermediate and inhibition of peptidoglycan synthesis in an in vitro system using a particulate fraction from Bacillus megaterium KM with UDP-MurNAc-[3H]pentapeptide and cold UDP-GlcNAc or cold UDP-MurNAc-pentapeptide and UDP-[3H]GIcNAc as substrates. At higher concentrations of azureomycin B (over 100 μg/ml), lipid intermediate accumulation was also inhibited.
When particulate fraction from Escherichia coli Y-10 and UDP-[14C]GlcNAc and cold UDP-MurNAc-pentapeptide were used, accumulation of lipid intermediate and inhibition of peptidoglycan synthesis were also observed.
These results indicate that the primary target of azureomycin B is the transfer of the disaccharide peptide unit (GlcNAc-MurNAc-pentapeptide) from lipid-bound precursor to acceptor.View full abstractDownload PDF (399K) -
Takashi KAGEYAMA, Kenji TAKAHASHI1980 Volume 88 Issue 2 Pages 571-582
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon activation of human pepsinogen A at pH 2.0 in the presence of pepstatin, an intermediate form was generated together with pepsin A. This activation intermediate could be separated from pepsinogen A and pepsin A by DE-32 cellulose chromatography at pH 5.5. It had a molecular weight intermediate between those of pepsinogen A and pepsin A, and contained about half the number of basic amino acid residues in pepsinogen A. It had phenylalanine as the amino(N)-terminal amino acid, and was deduced to be generated by release of the N-terminal 25 residue segment from pepsinogen A. Amino acid sequence determination of the N-terminal portions of pepsinogen A and the intermediate form enabled us to elucidate the entire amino acid sequence of the 47-residue activation peptide segment as follows:
1Ile-Met-Tyr-Lys-V al-Pro-Leu-Ile-Arg-10Lys-Lys-Ser-Leu-Arg-Arg-Thr-Leu-Ser-Glu-20Arg-Gly-Leu-Leu-Lys-Asp-Phe-Leu-Lys-Lys-30His-Asn-Leu-Asn-Pro-Ala-Arg-Lys-Tyr-Phe-Pro-Gln-Arg-Lys-Ala-Pro-Thr-47Leu.
On the other hand, upon activation of pepsinogen A at pH 2.0 in the absence of pepstatin, cleavage of the activation segment occurred at several additional bonds. In addition, upon activation both in the presence and in the absence of pepstatin, an additional activation intermediate, designated pepsin A', was formed in minor quantities. This form was identical with pepsin A, except that it had an additional Pro-Thr-Leu sequence preceding the N-terminal valine of pepsin A.View full abstractDownload PDF (1849K) -
The Structures of Three Novel Gangliosides from the Fat Body of the FrogMasako OHASHI1980 Volume 88 Issue 2 Pages 583-589
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree unusual gangliosides were isolated from frog fat body, and their structures were characterized. The carbohydrate structures were elucidated by determination of the molar ratio of the individual monosaccharides, methylation analysis, galactosidase treatment, and chromium trioxide oxidation studies. The following novel structures are proposed:
Galα1_??_3Galβ1_??_3Ga1NAcβ1_??_4Ga1(3_??_2αNeuAc)β1_??_4Glcβ1_??_lCer; Galβ1_??_3Galα1_??_3Galβ1_??_3GalNAcβ1_??_4Gal(3-2αNeuAc)β1_??_4Glcβ1_??_1Cer; Galα1_??_3Galβ1_??_3Galα1_??_3Galβ1_??_3GalNAcβ1_??_4Gal(3_??_2αNeuAc)β1_??_4Glcβ1_??_1Cer.View full abstractDownload PDF (1544K) -
Kazuhiro KOHAMA1980 Volume 88 Issue 2 Pages 591-599
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mode of calcium binding to cardiac and fast skeletal troponins was studied at various proton and Mg ion concentrations, and the results thus obtained were compared with the superprecipitation of cardiac and fast skeletal myosin B under specified conditions. The high and low affinity sites of cardiac troponin showed many properties in common with those of fast skeletal troponin.
1. In low Mg ion concentrations the high affinity sites of both cardiac and fast skeletal troponins showed extraordinarily high affinity for Ca ions. They also exhibited very strong affinity for Mg ions in the virtual absence of Ca ions. These findings are discussed in relation to conformational changes of troponin C.
2. With increase in Mg ion concentration the extraordinarily high affinity for Ca ions gradually disappeared. The dissociation constant of the high affinity sites for Mg ions at millimolar Mg ion concentrations of cardiac and skeletal troponins were 2.6×10-3M, and those of the low affinity sites were 1-2×10-2M. These observations are compatible with the Mginduced shift of the relationship between pCa and contractile response (Ebashi & Endo (1968) Prog. Biophys. Mot. Biol. 18, 123-183).
3. In the presence of millimolar Mg ion concentrations, fast skeletal troponin showed different affinities for Ca ions at different pHs (6.2, 6.8, and 7.4), exhibiting higher affinity at higher pH. In the case of cardiac troponin, no difference in the affinity for Ca ions could be detected at different pHs in the presence of Mg ions, but in the absence of Mg ions a clear difference was seen at every pH.
4. Superprecipitation of cardiac and fast skeletal myosin B at low concentrations of Mg ions and ATP was regulated by Ca ions in the concentration range between 10-9-10-7M, where only the high affinity sites of troponin were involved in Ca binding. This observation further substantiates the view that the high affinity sites can directly regulate the contractile processes (Kohama, K. (1979) J. Biochem. 86, 811-820).View full abstractDownload PDF (650K) -
Tateo YAMANAKA, Seiji INOUE, Toshiki HIROYOSHI1980 Volume 88 Issue 2 Pages 601-604
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structural difference between larval and adult cytochromes c of the housefly, Musca domestica was studied. The two cytochromes differed in electrophoretic behavior and amino acid composition from each other. Peptide maps prepared from the tryptic digests of the two cytochromes were also different from each other in several regions. Amino acid analyses of the peptides separated on the maps showed that the primary structure differed between the two cytochromes in at least 5 positions.View full abstractDownload PDF (281K) -
Masaaki KURODA, Tomoh MASAKI1980 Volume 88 Issue 2 Pages 605-608
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExtractability of 42, 000 dalton components (42K component) from myosin-removed myofibrils (called I-Z-I segments in this paper) under low-salt conditions was compared at pH 6.5 and pH 8.0.
Kinetic and isoelectric focusing analysis of the 42K components revealed that the 42K component extracted at pH 8.0 was actin, which depolymerized from the end of the I-filame, lts of the I-Z-I segments. On the other hand, isoelectric focusing of the 42K component e _??_ tracted at pH 6.5 indicateds two protein bands. The relative mobility of the minor band was virtually identical to that of actin. While the isoelectric point of the major band was more acidic than that of skeletal muscle actin. Therefore, it is concluded that there are at least two kinds of 42, 000 dalton components in the myosin-removed myofibrils.View full abstractDownload PDF (953K) -
Kazuya TANIGUCHI, Kuniaki SUZUKI, Junichi SHIMIZU, Shoichi IIDA1980 Volume 88 Issue 2 Pages 609-612
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNa+, K+-ATPase [EC 3. 6. 1. 3] from pig kidneys was treated with the fluorescent reagent N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) in the presence of KCI. The resultant preparation showed 70% of the activity with only a small change in the apparent affinity for ligands of the enzyme. The addition of Na+ to the treated preparation induced a -2.1±0.1% change of the total fluorescence intensity observed in the absence of Na+. Further addition of both Mg2+ and ATP transiently increased the fluorescence to +0.5±0.1%. After the exhaustion of ATP, the fluorescence decreased to -3.1±0.1%. This cycle can be repeated by the readdition of ATP but not by ADP. Ouabain inhibits the fluorescence change. The ligands used reduced the fluorescence intensity as follows: Mg2++Na++ATP≈K+, none, Mg2+≈ATP, Na++ATP, Na+≈Na++Mg2+, Na++Mg2++ADP≈Na++ADP. The data indicate the presence of multiple conformational states of the enzyme.View full abstractDownload PDF (280K) -
Keiko HATAKEYAMA, Masahiko HIRAMATSU, Naomi MINAMI1980 Volume 88 Issue 2 Pages 613-615
Published: August 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptozotocin diabetes strongly reduced the N-acetyl-β-glucosaminidase activity in the submaxillary glands of mice. A reciprocal change was observed between the enzyme activity and blood sugar in diabetes. Insulin treatment of the diabetic mice returned the reduced activity to the normal level. Isoelectric focusing analysis showed that the submaxillary gland enzyme was composed of two isozymes having isoelectric points (pI) of 4.8 and 8.8. Only the pI 8.8 isozyme was affected by the diabetes.View full abstractDownload PDF (208K) -
1980 Volume 88 Issue 2 Pages 617
Published: 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (12K)
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