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Volume 82 (1977)
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Volume 82, Issue 3
Displaying 1-34 of 34 articles from this issue
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I. Comparison of the Microsomal, Golgi, and Lysosomal β-GlucuronidasesHiroshi TSUJI, Noriko HATTORI, Torao YAMAMOTO, Keitaro KATO1977 Volume 82 Issue 3 Pages 619-636
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study the role of the Golgi complex in the formation of lysosomes, a resonably pure Golgi fraction was prepared from rat livers. Enzyme profile and morphology of the Golgi fraction were studied. β-Glucuronidase [EC 3. 2. 1. 31] found in the Golgi fraction was proved to be indigenous to the Golgi complex by hypotonic-shock treatment of various subcellular fractions. Treatment of a low concentration of deoxycholate (DOC) revealed that about 65% of the total β-glucuronidase in the Golgi fraction was in the Golgi contents and about 35% of the enzyme was bound to the Golgi membrane. Detergent-dependent release of β-glucuronidase from the microsomal fraction also indicated that about 60% of the total glucuronidase in this fraction was in the cisternal lumen and the remaining 40% was bound to the membrane of the endoplasmic reticulum. Three β-glucuronidases solubilized from the microsomal, Golgi, and lysosomal fractions were compared by polyacrylamide gel electrophoresis and isoelectric focusing. These techniques indicated the resemblance of the Golgi β-glucuronidase to the lysosomal enzyme and the difference of the microsomal β-glucuronidase from the other two. In particular, gel isoelectric focusing emphasizes the similarity between the Golgi and lysosomal β-glucuronidases. This technique resolved the lysosomal β-glucuronidase into thirteen activity bands in an acidic pH range, in which four bands (L5, L7, L9, and L11) were more intensely stained. The Golgi β-glucuronidase exhibited six activity bands, of which G1, G2, G3, and G4 predominated and corresponded to L5, L7, L9, and L11, respectively. The microsmal β-glucuronidase showed five activity bands in a neutral pH range. There were no significant differences between the three enzymes with respect to Km and pH optimum. However, the microsomal β-glucuronidase differed significantly in heat stability from the other two enzymes. From the resemblance of the Golgi β-glucuronidase to the lysosomal enzyme, it is surmised that β-glucuronidase in the Golgi complex may be subsequently transferred into the lysosomes. If the newly synthesized enzyme at the site of membrane-bound ribosomes enters the cisternal lumen of the endoplasmic reticulum and is transferred into the lysosomes via the Golgi complex, the major transformation of the enzyme may occur in the Golgi complex, and the minor one in the lysosomes.View full abstractDownload PDF (3154K) -
II. Intracellular Transport of β-GlucuronidaseHiroshi TSUJI, Keitaro KATO1977 Volume 82 Issue 3 Pages 637-644
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to trace the route of intracellular transport of lysosomal enzymes, β-glucuronidase [EC 3. 2. 1. 31] in the endoplasmic reticulum and the lysosomes was purified from rat liver and the sequence of appearance of the newly synthesized enzyme was followed with respect to these organelles after labeling the enzyme with L-[3H]leucine. β-Glucuronidase was purified from the microsomal fraction and the mitochondrial-lysosomal fraction by organic solvent fractionation, gel filtration, isoelectric focusing, and acrylamide gel-sucrose gradient electrophoresis. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel. After the injection of the labeled amino acid, the microsomal, β-glucuronidase rose to a peak of specific radioactivity at 9h. The activity then decreased up to 18h and the decreased activity was maintained up to 24h. Between 9 and 18h after administration, when the specific radioactivity of the microsomal enzyme was decreasing, an increase in the activity of the lysosomal enzyme was observed and the maximum value was maintained up to 24h. The time-course of the appearance of labeled β-glucuronidase in these organelles after administration of the labeled amino acid suggests that the newly synthesized enzyme is transported from the endoplastic reticulum to the lysosomes, probably through the Golgi complex. As the appearance of the peak of radioactivity of β-glucuronidase in the microsomes was delayed, an alternate pathway of enzyme transport is also discussed.View full abstractDownload PDF (1141K) -
Motonori OHNO, Jun-ichiro SARUWATARI, Masako WATANABE1977 Volume 82 Issue 3 Pages 645-651
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe susceptible bonds of substrates of the type PABz-Glyn-Arg-R (R=OCH3, n=0, 1, 2; R=OH, n=1, 2) to papain [EC 3. 4. 22. 2] have been identified. PABz-Arg-OMe was found to be approximately 30 times more reactive than benzoylarginine ethyl ester, due to a Km(app) value 25 times smaller. The finding that PABz-Gly2-Arg-OMe was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Arg-OMe indicated that the S4 subsite of the enzyme is capable of accommodating a large hydrophobic group. Other substrates were cleaved at the peptide bond one residue removed from the PABz group, in accord with the well-known fact that the S2 subsite interacts preferentially with a hydrophobic P2 residue. A negative charge on the substrate at the P2' or P3' position greatly decreases the acylation rate and also the noncovalent binding affinity. The hydrolysis of PABz-Arg-OMe and PABz-Gly2-Arg-OMe was little affected by the acridine dye profiavine.View full abstractDownload PDF (544K) -
III. Changes in the Distribution of Exposed Lysine Residues among Subfragments with Change in Enzymatic StateTaibo YAMAMOTO, Yuji TONOMURA1977 Volume 82 Issue 3 Pages 653-660
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSR vesicles were allowed to react for 15min at pH 8.0 and 0°C with an impermeant reagent, TBS, fixing the Ca2+-dependent ATPase [EC 3. 6. 1. 3] in any one of the three enzymatic states: MgE, MgEATP, and E_??_P. The TNP-SR was digested with trypsin, and three major subfragments of the ATPase (I, II, and III) with molecular weights of about 50, 000, 32, 000, and 22, 000 daltons were separated by SDS-polyacrylamide gel electrophoresis. The amounts of TNP incorporated into these subfragments were measured after eluting them from gels with 1% SDS.
When SR was incubated with TBS in the enzymatic state MgE, about 1, 0.5, and 0.5 mol of TNP were incorporated for each mole of subfragments I, II, and III, respectively. About 0.5, 0.5 and 0 mol of TNP were incorporated for each mole of subfragments I, II, and III, respectively, when the enzymatic state was fixed at MgEATP. On the other hand, about 1.5, 1, and 1 mol of TNP were incorporated for each mole of subfragments I, II, and III, respectively, in state E_??_P.
SR was further purified by sucrose density gradient centrifugation, and was digested with trypsin. The SDS-gel electrophoretograms of tryptic digests changed markedly with change in the enzymatic state of the ATPase, MgE, MgEATP or E_??_P.
Based on these results, together with those on the molecular mechanism of coupling of the ATPase reaction with Ca2+ transport, a diagrammatic model explaining the functional movements of the ATPase molecule coupled with elementary steps of the ATPase reaction is proposed.View full abstractDownload PDF (585K) -
Makoto IWATA, Hayao IDE, Tadao TERAO, Toshiaki OSAWA1977 Volume 82 Issue 3 Pages 661-669
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe major glycoproteins which bind various lectins (Bauhinia purpurea, Lens culinaris, Pisum sativum, Ricinus communis, and wheat germ hemagglutinins) were isolated from splenic lymphocytes of DD-mice. The surface of the cells was radioiodinated using the enzyme lactoperoxidase and the crude membranes (microsomal fraction) were isolated from the radioiodinated cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on affinity adsorbents prepared by coupling the lectins to activated Sepharose 4B. The glycoproteins specifically eluted with haptenic sugars from the affinity adsorbents were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The mitogenic lectins (P. sativum, L. culinaris) and R. communis hemagglutinin, which preferentially interact with the sugar chains of the type found in serum glycoproteins (serum glycoprotein-type sugar chains), bind strongly membrane glycoproteins with molecular weights ranging from 80, 000 to 20, 000, whereas the other lectins, which can interact with sugar chains such as those of mucins (mucin-type sugar chains), bind membrane glycoproteins with molecular weights higher than 100, 000. To characterize the receptor sites for mitogenic lectins on the surface of lymphocytes, the membrane glycoproteins of C3H/He-mouse splenic lymphocytes, which were specifically eluted from a column of P. sativum-Sepharose 4B, were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, and the histocompatibility-2-complex proteins (H-2D, H-2K, and Ia antigens) were found to be major receptor sites.View full abstractDownload PDF (695K) -
Specificity of Glycerophosphate, 1-Acylglycerophosphate and 2-Acylglycerophosphorylcholine Acyltransferase SystemsKeizo WAKU, Fumie HAYAKAWA, Yasuo NAKAZAWA1977 Volume 82 Issue 3 Pages 671-677
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The activities for the acylation of glycerol 3-phosphate, 1-acyl-sn-glycerol 3-phosphate and 2-acyl-sn-glycero-3-phosphorylcholine with several fatty acyl-CoA's were estimated with rabbit sarcoplasmic reticulum.
2. The highest incorporation rate was observed at the transfer of linoleate to glycerol 3-phosphate, and linolenate was incorporated most slowly. In the acylation of 1-acyl-sn-glycerol 3-phosphate, the incorporation rate of linolenate was highest, with that of linoleate coming next, and other fatty acids were incorporated at practically the same lower rate. However, strict substrate specificity was not observed in the synthesis of phosphatidic acid in rabbit sarcoplasmic reticulum.
3. The highest rate was observed in the incorporation of palmitate at the 1-position of 2-acyl-sn-glycero-3-phosphorylcholine, suggesting the participation of this enzyme in the regulation of fatty acid composition at the 1-position of phosphatidylcholine of rabbit sarcoplasmic reticulum.
4. Since the specific activity of 1-acyl-sn-glycero-3-phosphorylcholine acyltransferase is about 10 times higher than that of 2-acyl-sn-glycero-3-phosphorylcholine acyltransferase, the fatty acid pattern of phosphatidylcholine of rabbit sarcoplasmic reticulum may be controlled by 1-acyl-sn-glycero-3-phosphorylcholine acyltransferase to a larger extent than by 2-acyl-sn-glycero-3-phosphorylcholine acyltransferase.
5. Thus, the specificities of 1-acyl-sn-glycerol 3-phosphate and 2-acyl-sn-glycero-3-phosphoryl-choline acyltransferases are largely consistent with the general fatty acid patterns at the 1- and 2-positions of phospholipids. However, the fatty acid composition of phosphatidylcholine does not completely reflect the specificities of these enzymes.View full abstractDownload PDF (497K) -
Hiroko YABUKI, Koichi IWAI1977 Volume 82 Issue 3 Pages 679-686
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSoluble chromatin was fragmented by deoxyribonuclease I [EC 3.1.4.5] or II [EC 3.1.4.6] digestion or sonication, followed by 0.7M NaCl treatment, which preferentially removed DNA fragments and histones from the fragmented chromatin, but not from intact chromatin, leaving an insoluble fraction of DNA-histone-nonhistone protein complex without appreciable redistribution of the chromatin components. Fragmentation of DNA and concentration of nonhistone proteins in the fraction were most effective with deoxyribonuclease I under the conditions used. Thus, only 1% of the DNA in transcriptionally inactive chicken erythrocyte chromatin was recovered, together with 2% of the total histone and 50-100% of the total nonhistone protein. Separate experiments showed that this DNA fraction was among the double-stranded segments having an average size of 210 base pairs and was associated with histone components responsible for the nucleosome conformation as well as most of the nonhistone protein components in the original chromatin. From transcriptionally active chicken liver or calf thymus chromatin, however, 5-10% of the total DNA, 20-40% of the histone, and 80-10% of the nonhistone protein were recovered in the corresponding fraction. These results may reflect the native states and template activities in situ of various chromatins.View full abstractDownload PDF (1220K) -
Naoko ARAI, Ken-ichi ARAI, Yoshito KAZIRO1977 Volume 82 Issue 3 Pages 687-694
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation of a binary complex between the polypeptide chain elongation factor G (EF-G) and guanine nucleotides was demonstrated by hydrophobic probes and equilibrium studies using gel filtration (Arai, N., Arai, K., & Kaziro, Y. (1975) J. Biochem. 78, 243-246). In the present communication, the interaction of EF-G with guanine nucleotides is investigated in more detail using equilibrium dialysis and a hydrophobic probe, 1-anilino-8-naphthalene sulfonate (ANS).
EF-G possesses one binding site for guanine nucleotide per mole of protein, and the dis-sociation constants for GTP and GDP determined by equilibrium dialysis were 1.0×10-4M, and 5.0×10-5M, respectively. The fluorescence intensity of ANS elicited in the presence of EF-G was markedly diminished by the addition of GTP, and, to a lesser extent, by the addition of GDP. The dissociation constants of EF-G•GTP and EF-G•GDP estimated by fluorometric titration were 1.4×10-5M, and 1.1×10-5M, respectively. The nonhydrolyzable analogues of GTP, GMP-P(NH)P, and GMP-P(CH2)P, showed similar quenching of fluorescence of ANS, and the dissociation constants of EF-G•GMP-P(NH)P and EF-G•GMP-P(CH2)P were calculated as 4.3×10-5 M and 5.7×10-5M, respectively. The number of binding sites for ANS of EF-G, EF-G•GDP, and EF-G•GTP were estimated by equilibrium gel filtration as 5, 5, and 4, respectively.
The difference in fluorescence emission spectra and the number of binding sites for ANS of EF-G•GTP and EF-G•GDP suggests a conformational difference between these two forms of EF-G.View full abstractDownload PDF (590K) -
Naoko ARAI, Ken-ichi ARAI, Shun NAKAMURA, Yoshito KAZIRO1977 Volume 82 Issue 3 Pages 695-702
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties and function of sulfhydryl groups in the polypeptide chain elongation factor G (EF-G) have been studied by titration with p-chloromercuri [14C] benzoate, or 5, 5'-dithiobis (2-nitrobenzoic acid), and by assessing the inhibitory effect of N-ethylmaleimide on its activity. EF-G contained three sulfhydryl groups per mole of protein, of which only one was reactive under native conditions. The other two were nonreactive and could be titrated only after complete denaturation of the protein. The activity to catalyze the uncoupled GTPase reaction as well as the ability to form a ternary complex involving ERG, guanine nucleotides, and ribosomes was completely inactivated by treatment of EF-G with N-ethylmaleimide.
The reactive sulfhydryl group was not required for interaction with guanine nucleotides, but was essential for binding to ribosomes. The binary complex involving EF-G and GDP could be isolated after treatment of EF-G with sulfhydryl reagents. The sulfhydryl group of EF-G was not protected by guanine nucleotides alone, but a marked protection was afforded by addition of ribosomes and GMP-P(CH2)P.
The reactivity of the sulfhydryl group of EF-G was modulated by interaction with guanine nucleotides. The kinetic studies indicated that reactivity toward N-(1-anilinonaphthyl-4)-maleimide was reduced by GDP and, to a lesser extent, by GTP.
These results, together with the previous report on the spin-label probes (Arai, N., Arai, K., Maeda, T., Ohnishi, S., and Kaziro, Y. (1976) J. Biochem. 80, 1057-1065) indicate that (1) the reactive sulfhydryl group of EF-G is required for interaction with ribosomes, and (2) its reactivity is modulated by guanine nucleotide-induced conformational transitions occurring near the reactive protein sulfhydryl.View full abstractDownload PDF (561K) -
Kazuo MOTOYOSHI, Kentaro IWASAKI1977 Volume 82 Issue 3 Pages 703-708
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolypeptide chain elongation factor-1βγ (EF-1βγ), an eukaryotic counterpart to bacterial EF-Ts, has been purified to a homogeneous state, and shown to have a molecular weight of about 90, 000. It consisted of unequal subunits with molecular weights of 30, 000 (EF-1β) and 55, 000 (EF-1γ). Thus, separation of these subunits was attempted by gel filtration of EF-1βγ treated with guanidine hydrochloride on a column of Sephadex G-200 in the presence of urea. After removing urea by dialysis, the enzymatic activities of the separated subunits were examined by means of the following three reactions, all of which have previously been shown to be stimulated by EF-lβγ; i) exchange of GDP bound to EF-1α with exogenous GTP, ii) EF-1α-dependent binding of Phe-tRNA to ribosomes, and iii) poly (U)-dependent polyphenylalanine synthesis. Under the standard conditions, EF-1β stimulated all of these reactions to the same level as EF-1βγ when the amount of the former was adjusted to be onethird of that of the latter, or the molar concentrations of these factors were the same. This suggests that the effect of EF-1βγ on these reactions could be accounted for that of the EF-1β moiety in the complex. However, when the concentrations of both magnesium acetate and KCI in the reaction mixture were increased, it was observed that EF-lβγ stimulated polyphenylalanine synthesis much more effectively than EF-1β. The amino acid composition of EF-1β as well as EF-1γ was determined.View full abstractDownload PDF (436K) -
Toshihiro TSUDZUKI, Toshihiro FUJII, Ryo TANAKA1977 Volume 82 Issue 3 Pages 709-717
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. As a part of studies on the mechanism by which catecholamines are released from the nerve terminals, the synaptic vesicle fraction was isolated from bovine caudatolenticular nuclei and thalamus by differential centrifugation essentially according to the method of Kadota and Kadota (17).
2. Further centrifugation on a sucrose density gradient of the synaptic vesicle fraction by the method of Whittaker et al. (1) yielded white materials on the upper portion of 0.4M sucrose, which consisted of vesicles averaging 600-800 A in diameter, and did not show Mg2+-dependent ATPase activity. On the other hand, the denser materials centering on 0.6M sucrose, consisting of a mixture of microsomes and synaptic vesicles of 400-500 A diameter, showed an ATPase activity activated by either Mg2+ or Ca2+ but not inhibited by ouabain.
3. The white materials on 0.4M sucrose were almost free of mitochondria, but they contained a large amount of non-heme iron, as reported elsewhere (2). Furthermore, the protein components analyzed on SDS-polyacrylamide gels were similar to those already reported for purified synaptic vesicles (3).
4. Based on these results, the white materials were assumed to be synaptic vesicles devoid of Mg2+-dependent ATPase activity.View full abstractDownload PDF (1237K) -
Shinji YOKOYAMA, Toshio MURASE, Yasuo AKANUMA1977 Volume 82 Issue 3 Pages 719-724
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLecithin: cholesterol acyltransferase (LCAT) was more highly activated by apolipoprotein A-I (apoA-I) with dimyristoyl phosphatidyicholine (DMPC) than with dilinoleoyl phosphatidylcholine (DLPC) when lipid dispersion of cholesterol and each phosphatidylcholine was used as a substrate. When the enzyme reactions were activated by whole apolipoproteins of high density lipoproteins (HDL), DLPC was more available to the LCAT reaction than DMPC with high concentrations of apoHDL in an incubation mixture. However, no detectable enzyme reaction was observed with dipalmitoyl phosphatidyicholine (DPPC) under both conditions. On the other hand, all of these phosphatidylcholines acted as substrates of LCAT when they were incorporated into HDL coupled to Sepharose. The order of their relative reactivities to cholesterol was DMPC, DPPC, and DLPC under the conditions used.View full abstractDownload PDF (429K) -
Takashi SHIMAKATA, Yoko FUJITA, Takashi KUSAKA1977 Volume 82 Issue 3 Pages 725-732
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insensitive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of β-oxidation, was not functional in the elongation system.View full abstractDownload PDF (609K) -
Active Site MappingTakehiko WATANABE, Esmond E. SNELL1977 Volume 82 Issue 3 Pages 733-745
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTryptophanase [TPase, L-tryptophan indole-lyase, EC 4. 1. 99. 1] from Escherichia coli B was studied extensively with respect to its interaction with various amino acids and their analogs. Several new substrates were found; these include β-chloro-L-alanine, L-cysteine sulfinate, S-benzyl-L-cysteine, O-benzyl-L-serine, L-threonine, L-allothreonine, and the two β-methyl-L-tryptophans. Formation of S-hydroxyethyl-L-cysteine from L-serine and 2-mercaptoethanol was also demonstrated. Most amino acids tested, including these substrates, were competitive inhibitors of the TPase reaction with L-tryptophan as the substrate. The K1 values differed so markedly among various amino acids that it was possible to draw conclusions concerning the topography of the active site of TPase. The indole ring or other electronegative group on the β-carbon atom, together with a carboxyl group on the α-carbon atom, is necessary for oriented binding to the active site of TPase; a free α-amino group is also required for substrate activity, but does not contribute greatly to the binding interactions.
Most amino acids and their analogs induced an absorption peak near 500nm, previously assigned to quinoidal structure formed by labilization of the α-proton of the enzyme-bound Schiff's base between PLP and amino acid. Amino acids that have an asymmetric carbon at the β-position (L-threonine, L-allothreonine, L-isoleucine, , β-methyl-L-tryptophan) and some other amino acids (L-valine, phenylglycine, glycine) did not evoke this 500nm peak.
Bromopyruvate proved to be a very potent inactivator of TPase. Pyridoxal 5'-phosphate completely prevented this inactivation, but inhibitor amino acids did not. One SH group per subunit of TPase was blocked by treatment with bromopyruvate. The cysteine residue that reacts is probably located in the peptide T-18-1 isolated by Kagamiyama et al. [J. Biol. Chem. 247, 1571-1575 (1972)]. TPCK inactivated TPase in a manner similar to bromopyruvate.View full abstractDownload PDF (969K) -
I. Effects of Temperature, Ionic Strength, Divalent Metal Ions, and NucleotidesKeiichi YAMAMOTO, Takamitsu SEKINE1977 Volume 82 Issue 3 Pages 747-752
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLight chains of skeletal muscle myosin were studied through the reactivity of their SH groups with a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM). The experiments were carried out by reacting the reagent with myosin for a short time and measuring the amounts of reacted dye by fluorometry after separating light chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The two classes of light chains, alkali light chains and DTNB light chain, were clearly distinguished by their manner of reactivity change, and differences in their environment and in their function were suggested. Although we found that the SH groups of the DTNB light chain were susceptible to very low concentrations of Mg ions (of the order of 10-5M), we could not observe Ca2+-induced conformational change by our technique.
We also estimated the stoichiometry of light chains in skeletal muscle myosin to be 1.37 mol alkali light chain 1, 1.95 mol of DTNB light chain and 0.77 mol of alkali light chain 2 per mole of myosin from the total amounts of our reagent that reacted with each light chain.View full abstractDownload PDF (439K) -
Shoko OHTANI, Tsutomu OKADA, Hajime YOSHIZUMI, Hiroyuki KAGAMIYAMA1977 Volume 82 Issue 3 Pages 753-767
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurothionin (a lethal protein for brewer's yeast) obtained from wheat flour (Triticum vulgare, Manitoba No. 3) was separated into two active fractions, A and B, by CM-cellulose column chromatography. The main component, purothionin A, was purified to a homogeneous state as shown by polyacrylamide gel disk electrophoresis and isoelectric focusing. The molecular weight of purothionin A was estimated by the sedimentation equilibrium method to be 11, 300 in its native state, 5, 500 in 6M guanidine HCl and after its modification with succinic anhydride. Chemical studies including the determination of N- and C-terminal amino acid residues and amino acid sequence analyses using a combination of automatic Edman degradation and tryptic digestion of purothionin A revealed its dimeric structure composed of nonidentical and closely similar polypeptide chains. Two distinct polypeptides were isolated by DE-52 column chromatography after succinylation. Based on the amino acid composition of each polypeptide, it was possible to align all the tryptic peptides of purothionin A without any ambiguities, establishing the complete primary structures of the purothionin A subunits.
Lys-Ser-Cys-Cys-_??_-Ser-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Lys-Ser-Cys-Cys-_??_-Ser-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Cys-Arg-Ala-Arg-Gly-Ala-Gln-Lys-Leu-Cys-Ala-_??_-Val-Cys-Arg-Cys-Arg-Ala-Arg-Gly-Ala-Gln-Lys-Leu-Cys-Ala-_??_-Val-Cys-Arg-View full abstractDownload PDF (2334K) -
Koyu HON-NAMI, Tairo OSHIMA1977 Volume 82 Issue 3 Pages 769-776
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA c-type cytochrome, cytochrome c-552, from a soluble fraction of an extreme thermophile, Thermus thermophilus HB8, was highly purified and its properties investigated. The absorption peaks were at 552, 522, and 417nm in the reduced form, and at 408nm in the oxidized form. The isoelectric point was at pH 10.8, the midpoint redox potential was about +0.23 V, and the molecular weight was about 15, 000. The cytochrome c-552 was highly thermoresistant. The cytochrome reacted rapidly with Pseudomonas aeruginosa nitrite reductase [EC 1. 9. 3. 2], but slowly with bovine cytochrome oxidase [EC 1. 9. 3. 1], yeast cytochrome c peroxidase [EC 1.11.1.5], or Nitrosomonas europaea hydroxylamine-cytochrome c reductase [EC 1. 7. 3. 4].View full abstractDownload PDF (524K) -
Toyoki KATO, Yuji TONOMURA1977 Volume 82 Issue 3 Pages 777-782
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA myosin B-like protein was extracted from the alga Nitella flexilis. SDS-polyacrylamide gel electrophoresis revealed the presence of myosin heavy chain and actin as the main components.At high ionic strength, its ATPase [EC 3. 6. 1. 3] reaction was activated by EDTA or Ca2+ and inhibited by Mg2+. At low ionic strength, superprecipitation was induced by the addition of ATP.
Myosin was purified from Nitella myosin B. The molecular weight of the heavy chain of Nitella myosin, estimated by SDS-gel electrophoresis, was slightly higher than that of skeletal muscle myosin. At low ionic strength, Nitella myosin aggregated to form bipolar filaments about 0.2μm long. At high ionic strength, its ATPase reaction was activated by EDTA or Ca2+, and inhibited by Mg2+. The Mg2+-ATPase reaction of Nitella myosin was activated by skeletal muscle F-actin.View full abstractDownload PDF (1082K) -
V. Enzymatic Properties of Heads B and AAkio INOUE, Kei KIKUCHI, Yuji TONOMURA1977 Volume 82 Issue 3 Pages 783-800
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious amounts of ATP were added to a complex of F-actin with subfragment-1 of HMM (acto-S-1) in the presence of pyruvate kinase [EC 2. 7. 1. 40] and PEP as an ATP regenerating system, and S-1 was separated by ultracentrifugation into two fractions, i.e., S-1 bound to F-actin (precipitate) and S-1 dissociated from F-actin (supernatant). S-1 fractions with P1 burst sizes from 0.2 to 0.8 mol/mol were obtained. The burst sizes of all the fractions were almost equal to those calculated by assuming that S-1 preparations contained equimolar amounts of S-1B which showed a P1 burst of 1 mol/mol of S-1 and S-1A which showed no P1 burst.
The rates of the Mg2+-ATPase [EC 3. 6. 1. 3] reaction in the steady state of two S-1 fractions with burst sizes of 0.75 and 0.20 mol/mol of S-1 were almost equal at various KCl concentrations at various pH values. The pH dependence of the decomposition rate constant (k4) of the reactive myosin-phosphate-ADP complex, MAPDP, was similar to that of the Mg2+-ATPase reaction in the steady state (v0). The values of k4 were 2.5-4.9 times those of v0 at 0°C, although they were almost equal at 20°C. These results indicate that the Mg2+-ATPase activity of S-1B in the state °M is equal to that of S-1A.
Both the Ca2+-ATPase and EDTA-ATPase activities of S-1 fractions were proportional to the burst size. Furthermore, when CaCl2 or EDTA was added to HMMADPP or °HMM produced by the reaction of ATP with HMM in the presence of MgCl2, the steady-state rate of the Ca2+- or the EDTA-ATPase reaction was observed after a lag phase of 2-3min. Therefore, we concluded that only S-1B has the Ca2+-ATPase and EDTA-ATPase activities.
When the Mg2+-ITPase reaction was started by adding ITP to HMMADPP or °HMM at 20°C, the steady-state level of the Mg2+-ITPase reaction was observed after a lag phase of about 1.5min. This result was consistent with our previous report (Inoue et al. (1975) J. Biochem. 77, 1135-1145) that ITP is mainly hydrolyzed by S-1B at 20°C. On the other hand, the steady-state level of hydrolysis of 8-Br ATP was observed immediately after the reaction had been started by adding 8-Br ATP to HMMADPP or °HMM. This supported our previous conclusion (Takenaka et al. (1976) J. Biochem. 80, 1381-1392) that 8-Br ATP is hydrolyzed only by S-1A.
We confirmed the result of Hozumi ((1976) Eur. J. Biochem. 63, 241-247) that the size of the P1 burst decreased from 0.5 to about 0 mol/mol of myosin head on decreasing the temperature from 20°C to 0°C. The decrease in P1 burst size with decreasing temperature was attributed to a shift of the equilibrium of the step M2ITP_??_MIDPP to the left, because: (1) when excess HMM was mixed with [γ-32P]ITP at low temperature, most of the added [γ-32P]ITP remained unhydrolyzed at the completion of the initial burst, and the apparent rate constant of decay of [γ-32P]ITP was unaffected by the subsequent addition of unlabelled ITP at 2 s after the reaction had been started, and (2), when HMMIDPP was formed by mixing excess HMM with [γ-32P]ITP at 20°C and then the temperature was rapidly decreased to 4°C, the amount of TCA-32P1 decreased rapidly and markedly to a low level, as might be expected on the basis of an equilibrium shiftView full abstractDownload PDF (1281K) -
VI. ATP Hydrolysis, Shortening, and Tension Development of MyofibrilsToshiaki ARATA, Yasuo MUKOHATA, Yuji TONOMURA1977 Volume 82 Issue 3 Pages 801-812
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSATPase activities [EC 3. 6. 1. 3] of myofibrils were measured in 50mM KCl and 2mM MgCl2 at pH 7.8 in the presence of various concentrations of ATP and free Ca2+ ions, using pyruvate kinase [EC 2. 7. 1. 40] and phosphoenolpyruvate as an ATP-regenerating system. Shortening rates of myofibrils were determined by measuring the average lengths of sarcomeres after stopping the contraction by adding 15mM EDTA. Isometric tensions and ATPase activities of glycerol-treated muscle fibers fixed at the rest length were measured in 50mM KCl and 2mM MgCl2 at pH 7.8 in the presence of various concentrations of ATP and free Ca2+ ions at various temperatures, using creatine kinase [EC 2. 7. 3. 2] and creatine phosphate to regenerate ATP. The following results were obtained:
1. In the presence of a sufficient amount of ATP, the ATPase activity of myofibrils during shortening was 1.3-1.7s-1, while the activity decreased to 0.2s-1 after completion of the shortening. Glycerol-treated muscle fibers fixed at the rest length also showed a low ATPase activity, about 0.1s-1.
2. The ATPase activity of myofibrils during shortening reached a maximal level at about 20μM ATP. On the other hand, the shortening rate of myofibrils increased remarkably from 0.05 to 4.9μm/s, with an increase in the ATP concentration from 20 to 100μM.
3. The sarcomere shortening induced by hydrolysis of one molecule of ATP was calculated from the ATPase activity of myofibrils during shortening and the shortening rate. The value increased remarkably with increase in the ATP concentration, and reached more than 300 Å at 150μm ATP.
4. The shortening rate of myofibrils in the presence of EGTA exhibited substrate inhibition: it reached a maximal level of 0.0057μm/s at 5μm ATP, and decreased to 0.001μm/s at 100μM ATP.View full abstractDownload PDF (759K) -
Kikuko TAKEUCHI, Yuji TONOMURA1977 Volume 82 Issue 3 Pages 813-833
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin was prepared from bovine arterial smooth muscle, and the properties of its ATPase [EC 3. 6. 1. 3] reaction were compared with those of skeletal muscle myosin. A protein fraction, called Fraction-4 (Fr-4), was isolated from a crude arterial myosin preparation, and its effects on the ATPase activity of a complex of arterial myosin with skeletal muscle F-actin were examined. The following results were obtained.
1. The Ca2+- and EDTA-ATPase activities of arterial myosin were almost equal to those of skeletal muscle myosin. Furthermore, no significant differences were detected between arterial and skeletal muscle myosins in the rates of formation of reaction intermediates, M2ATP and MADPP, in the Mg2+-ATPase reaction. The amounts of initial P1-burst of these myosins were equal, i.e. about 1mol/4.8×105g.
2. The Mg2+-ATPase activity of arterial myosin was activated up to tenfold by adding skeletal muscle F-actin. However, the ATPase activity of reconstituted arterial actomyosin was much lower than that of skeletal muscle actomyosin.
3. The sensitivity of the ATPase activity of arterial myosin B to Ca2+ was unaffected by the addition of skeletal muscle F-actin, but was eliminated by the addition of skeletal muscle myosin. These results suggested that the Ca2+ regulating system of arterial muscle is linked with myosin.
4. Actomyosin reconstituted from purified arterial myosin and skeletal muscle F-actin showed a low ATPase activity with low Ca2+-sensitivity. When the ATPase reaction was started by adding ATP in the presence of the protein fraction Fr-4, actomyosin showed a high ATPase activity with high Ca2+-sensitivity.
5. Arterial myosin was incubated with ATP in the presence of Ca2+ ions and Fr-4, and then myosin was isolated. Myosin thus “modified” showed a high actin-activated ATPase activity. The high ATPase activity of “modified” actomyosin was unaffected by removal of Ca2+ ions even in the presence of Fr-4, but was markedly inhibited by removal of Ca2+ ions when the skeletal muscle tropomyosin-troponin system was added.View full abstractDownload PDF (3707K) -
Yoh OKAMOTO, Koichi YAGI1977 Volume 82 Issue 3 Pages 835-837
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAccording to the measurement of ESR spectrum, Ca2+ induced conformational changes of spin-labeled g2 bound to myosin in the presence of 1mM Mg2+. The half-maximal changes were observed at pCa 6.8 and at pCa 3.7. Spin-labeled phosphorylated g2 bound to myosin showed one transition at pCa 4.5, which shifted to pCa 6.5 after the dephosphorylation with E. coli alkaliphosphatase.View full abstractDownload PDF (192K) -
II. Role of β-146 and β-2 Histidines in the Alkaline Bohr EffectMasato OHE, Akihiko KAJITA1977 Volume 82 Issue 3 Pages 839-845
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe values of pKa for β-146 and β-2 histidines in human hemoglobin were determined by the mass spectrometric method using the hydrogen-deuterium exchange reaction. The effects of ligand binding and allosteric interaction on these pKa values were investigated.
The pKa values of β-146 histidine in the oxy- and deoxy-forms of the stripped hemoglobin were little affected by addition of inositol hexaphosphate (7.0 in the oxy- and 8.2 in the deoxy-form). Blocking of the reactive thiol group at β-93 cysteine decreased the pKa values of the amino acid remarkably in both the oxy- and deoxy-forms, whereas the difference in pKa between the oxy- and deoxy-forms (d pKa) remained unchanged. Values of the pKa in the tetramer of
the p-chain were found to be 7.4 in both the oxy- and deoxy-forms. The pKa value in methemoglobin was found to be 7.0.
Values of pKa of β-2 histidine on human hemoglobin were measured mass-spectrometrically after isolation of histidine as the methyl thiohydantoin derivative from deuterated hemoglobin by stepwise Edman degradation. From these measurements a pKa value of 6.9 for this histidine in both oxy- and deoxy-hemoglobin was found in the absence of inositol hexaphos-phate. The pKa value of the histidine in oxyhemoglobin was little affected by inositol hexaphosphate, while the value in deoxyhemoglobin was shifted to a higher value of 7.7 by inositol hexaphosphate.View full abstractDownload PDF (496K) -
Yoshimasa MORINO, Sumio TANASE, Takehiko WATANABE, Hiroyuki KAGAMIYAMA ...1977 Volume 82 Issue 3 Pages 847-852
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA procedure is described for the large-scale preparation of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase from pig heart. The procedure consists of: 1. extraction of both isoenzymes by heat treatment of homogenates prepared from minced and frozen heart muscle; 2. separation of each isoenzyme on a hydroxyapatite column; 3. purification of each isoenzyme by combinations of heat treatment, ammonium sulfate fractionation and chromatography on ion-exchange cellulose columns. Purified preparations of each isoenzyme thus obtained were homogeneous proteins as judged from their spectral properties and behavior on polyacrylamide gel electrophoresis. Using the present procedure, 1.2g of the cytosolic isoenzyme and 1.7g of the mitochondrial isoenzyme were obtained from 20kg of minced heart muscle.View full abstractDownload PDF (459K) -
Fumio MIAKE, Takao TORIKATA, Shuichi HANADA, Katsumi KOGA, Katsuya HAY ...1977 Volume 82 Issue 3 Pages 853-858
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMalic enzyme, which requires NADP+ as a coenzyme, was isolated and purified from pupae of the silkworm, Bombyx mori. The purified enzyme appeared homogeneous and had a molecular weight of 195, 000 on polyacrylamide gel electrophoresis. The optimum pH for the oxidative decarboxylation of malate, measured in terms of the increase of NADPH (MH activity) and CO2 (MC activity), was pH 7.5, while that for the decarboxylation of oxaloacetate measured in terms of the increase of CO2 (OC activity) was pH 4.6. Several differences between MH and OC activity were investigated.View full abstractDownload PDF (448K) -
Nobuhito SONE, Margareta K. LARSSTUVOLD, Yasuo KAGAWA1977 Volume 82 Issue 3 Pages 859-868
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe toxic effects of CH3HgCl on mitochondria of mammalian organs including human and rat liver were examined. [203Hg]CH3HgCl was bound mainly to mitochondrial proteins. The binding was not affected by the energy state of mitochondria. The state 3 respiration, oxidative phosphorylation and 32P1-ATP exchange reaction were inhibited by 10 to 50nmol of CH3HgCl per mg of mitochondrial protein, while NADH- and succinate-dehydrogenase and ATPase were more resistant to it. The difference spectrum of the treated mitochondria indicated that the point of inhibition was located after flavin and before cytochrome b. Mitochondrial swelling was induced by CH3HgCl, in accordance with previous morphological observations in vivo. The swelling, stimulation of ATPase and energy-dependent H+ extrusion caused by CH3HgCl were equally dependent on K+. Under these conditions, uptake of K+ by mitochondria was increased and the membrane potential was dissipated.
Unlike the case with other organomercuric compounds, transport of phosphate was not inhibited by CH3HgCl. When tested on liposomes, CH3HgCl itself was not lipid-soluble, as some organomercuric compounds are, and was not an uncoupler or a K+-carrier. It was concluded that protein bound CH3HgS-induced K+ uptake into mitochondria and the resulting loss of membrane potential was the major cause of uncoupling, though at higher concentrations, the electron transport system was also inhibited.View full abstractDownload PDF (720K) -
Chemical Transformation of the Active-Site Serine Residue of Streptomyces griseus Trypsin to a Cysteine ResidueHideyoshi YOKOSAWA, Setsuko OJIMA, Shin-ichi ISHII1977 Volume 82 Issue 3 Pages 869-876
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active-site serine residue of Streptomyces griseus trypsin was converted to a cysteine residue, and the product, thioltrypsin, was purified through two chromatographic steps with organomercurial-Sepharose and soybean trypsin inhibitor-Sepharose as specific adsorbents. The purified preparation of thioltrypsin was found to contain a single residue of cysteine and to react with almost equimolar amounts of normality titrants. It exhibited only traces of catalytic activity toward typical trypsin substrates such as Nα-tosyl-L-arginine methyl ester, whereas it retained some activity toward “active ester” substrates such as Nα-carbobenzoxy-L-lysine p-nitrophenyl ester. The activity was inhibited by sulfhydryl-blocking reagents, but no inhibition was observed by reagents reactive with the active hydroxyl group of serine proteases. Leupeptin, a natural trypsin inhibitor of peptidyl nature, also inhibited thioltrypsin. Some difference in the mode of leupeptin inhibition, however, was detected between trypsin and thioltrypsin. The bindings of small synthetic ligands and soybean trypsin inhibitor to thioltrypsin were compared with those to trypsin.View full abstractDownload PDF (645K) -
XXIV. Preparation and Properties of a Stable Water-Insoluble Polyacrylamide Derivative of Ribonuclease T1Hisashi ITO, Masaaki HAGIWARA, Kenji TAKAHASHI, Iwao ICHIKIZAKI1977 Volume 82 Issue 3 Pages 877-883
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibonuclease T1 [EC 3. 1. 4. 8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77%. of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of pH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37°C) or above 60°C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37°C after incubation in the range of pH 1 to 10 at 37°C, or after heating at 100°C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.View full abstractDownload PDF (485K) -
Michihiko TADA, Fumio OHMORI, Yasuharu NIMURA, Hiroshi ABE1977 Volume 82 Issue 3 Pages 885-892
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe partially purified a heat-stable protein factor (protein kinase modulator) from fresh bovine heart, which exhibited abilities to inhibit cardiac muscle adenosine 3': 5'-monophosphate (cyclic AMP)-dependent protein kinase [EC 2. 7. 1. 37] and to stimulate pulmonary guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase [EC 2. 7. 1. 37], and examined its effects on cyclic AMP-dependent protein kinase-catalyzed phosphorylation of cardiac microsomal protein “phospholamban” (MW=22, 000), which was previously proposed to serve as a regulator of calcium transport by cardiac sarcoplasmic reticulum (Tada, M., Kirchberger, M. A., & Katz, A. M. (1975) J. Biol. Chem. 250, 2640-2647). Incubation of cardiac microsomes, consisting mainly of sarcoplasmic reticulum, with cyclic AMP, protein kinase and [γ-32P] ATP in the presence of the modulator resulted in the inhibition of phosphorylation of the 22, 000-dalton protein component of the microsomal membranes. When microsomes were incubated under identical conditions with unlabeled ATP, the rate of oxalate-facilitated calcium uptake, which had been stimulated by cyclic AMP-dependent protein kinase, was found to decrease with increasing amounts of protein kinase modulator. The decrease in calcium uptake was closely correlated with the inhibition of phosphorylation of the 22, 000-dalton protein. These observations provide further evidence that the phosphorylation of the 22, 000-dalton protein is functionally related to calcium transport by cardiac sarcoplasmic reticulum of the heart, and thus support the previously proposed control mechanism of calcium transport.View full abstractDownload PDF (626K) -
Yoosuke YAMADA, Shizuo WATANABE1977 Volume 82 Issue 3 Pages 893-900
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA kinetic study of the ATPase reactions catalyzed by myosin and actomyosin was carried out by varying the concentrations of ATP and 2, 4-dinitrophenol (DNP). Mg-ATPase of myosin in the initial burst and that of actomyosin were both inhibited competitively by DNP. The dissociation constants for the DNP-myosin interaction (K1) were estimated to be very similar, that is, 4.2mM in the initial burst of ATP splitting, and 3.3mM for the actomyosin ATPase. It is therefore suggested that DNP acts at the same site when it inhibits the burst splitting of ATP and the actomyosin ATPase. In contrast, Mg, - Ca-, and EDTA-ATPase activities of myosin in the steady state were all affected uncompetitively by DNP. Moreover, the K1 value for Mg-ATPase of myosin in the steady state was found to be 31mM, which is much higher than those mentioned above for the initial burst and actomyosin ATPase. It is there-fore suggested that the site at which DNP acts to inhibit the burst splitting of ATP is different from the site at which DNP acts to affect Mg-, Ca-, and EDTA-ATPases in the steady state.View full abstractDownload PDF (395K) -
I. Synthesis and Activity of Nonapeptide Fragments of Soybean Bowman-Birk InhibitorNorikazu NISHINO, Haruhiko AOYAGI, Tetsuo KATO, Nobuo IZUMIYA1977 Volume 82 Issue 3 Pages 901-909
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo heterodetic cyclic nonapeptides, X-Cys-Thr-Lys-Ser-Asn-Pro-Pro-Gln-Cys-Y (Ia: X=Ac, Y=NH2; Ib: X=H, Y=OH), which correspond to residues 14-22 in the sequence of Bowman-Birk inhibitor, have been synthesized by Merrifield's solid-phase method. Inhibitory activities of la and Ib on tryptic hydrolysis of amide and ester substrates were examined. When Gly2-Lys-Gly3, and Tos-Arg-OMe were used as substrates, the values of I50 for the peptide Ia were calculated to be 3.6μM and 40μM, respectively. When Gly2-Lys-Gly3 was used as a substrate, the value of K1 was calculated to be 1.5μM. Ia was hydrolyzed slowly by trypsin, losing the inhibitory activity. When the Lys-Ser bond of la was cleaved with trypsin, the modified la could not be regenerated by trypsin. The linear peptide S, S'-dicarboxamido-methyl-Ia also was inactive and appeared to be a good substrate. Optical rotatory dispersion studies showed that the active fragments have characteristic conformations which were lost upon modification to inactive derivatives.View full abstractDownload PDF (655K) -
Ishtiaq MAHMUD, Yoshiaki MIURA1977 Volume 82 Issue 3 Pages 911-913
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAfter a period on protein-free diet and then starvation, high protein diet induces DNA synthesis in rat liver for a longer period than diet containing an equivalent amount of amino acids.The requirement for high protein diet is limited to an early part of the prereplicative periodand administration of the high protein diet later did not prolong DNA synthesis.View full abstractDownload PDF (200K) -
Shoji IDA1977 Volume 82 Issue 3 Pages 915-918
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAssimilatory nitrite reductase was purified 1, 700-fold with a yield of 22% from spinach leaves with a procedure involving ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex chromatography, gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous as shown by disc and SDS-gel electrophoresis with a specific activity (μmol NO2- reduced/min/mg protein) of 140.View full abstractDownload PDF (842K) -
Yoshiko OHNO-IWASHITA, Kazutomo IMAHORI1977 Volume 82 Issue 3 Pages 919-922
Published: September 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSContrary to the previous findings we found that a specific cleavage of 16S rRNA occurred when protein A, the active component of colicin E3, was interacted with isolated 30S ribosomal subunits at high concentrations. Necessary experiments to disprove the possibility of artefacts are described.View full abstractDownload PDF (1229K)
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