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Volume 92 (1982)
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Volume 92, Issue 5
Displaying 1-41 of 41 articles from this issue
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Mitsuharu SATO, Ikuo FUNAKOSHI, Kyozo HAYASHI, Ikuo YAMASHINA1982 Volume 92 Issue 5 Pages 1337-1345
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour forms of kallikrein, designated as I-IV, were isolated from hog pancreas autolysate mainly by chromatographies on Lysine-aminohexyl-Sepharose and on DEAE-Sepharose CL-6B, with yields of 65, 56, 59, and 41mg, respectively, from 10kg of the tissue. They were homogeneous on polyacrylamide gel electrophoresis, indistinguishable from eath other immunologically and had the same amino acid composition. Kallikreins I, II, and III contained carbohydrate, but kallikrein IV was essentially carbohydrate free. On reduction with mercaptoethanol, each of them produced two polypeptide chains with different molecular weights. The H (heavy) chains from kallikreins I and II were identical, designated as H1. It was a glycoprotein with apparent molecular weight of 21, 000, whereas the H chain from III and IV, designated as H2, had a molecular weight of 17, 000 and was regarded as an H1 chain devoid of carbohydrate. Likewise, the L (light) chain from kallikreins I and III, designated as L1, was a glycoprotein with an apparent molecular weight of 12, 000, whereas L2 from II and IV corresponded to L1 devoid of carbohydrate and had a molecular weight of 8, 500. Thus, kallikreins I-IV could be expressed as H1L1, H1L2, H2L1, and H2L2, respectively. They had comparable specific activities and Km values towards synthetic substrates. The isoelectric points of kallikreins I-IV were nearly the same, with values of 4.0-4.1.View full abstractDownload PDF (1856K) -
Toshiharu HASE, Hiroshi MATSUBARA, Georgina N. HUTBER, Lyndon J. ROGER ...1982 Volume 92 Issue 5 Pages 1347-1355
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequences of ferredoxins I and II from a blue-green alga, Nostoc strain MAC were determined. This alga is able to grow autotrophically in the light or heterotrophically in the dark. Analyses of tryptic peptides of Cm-proteins by conventional methods including solid-phase Edman degradation gave the complete amino acid sequences. Both molecules consisted of 98 amino acid residues and 34 amino acid differences including two deletions were found between the two. Comparing these sequences with those of ferredoxins from Chlorogloeopsis fritschii and Synechocystis 6714, which are also capable of growing under both conditions, showed that Nostoc strain MAC ferredoxin II had unique amino acids around the [2Fe-2S] cluster. This finding provides a structural basis for explaining the different chemical and functional properties of Nostoc strain MAC ferredoxin II reported in a previous paper (Hutson et al. (1978) Biochem. J. 172, 465-477).View full abstractDownload PDF (746K) -
Toshiharu HASE, Katsura INOUE, Hiroshi MATSUBARA, Myrtle M. WILLIAMS, ...1982 Volume 92 Issue 5 Pages 1357-1362
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of ferredoxin from Synechocystis 6714, a unicellular bluegreen alga, was determined by a combination of conventional methods. The ferredoxin was composed of 96 amino acid residues and lacked methionine and tryptophan. The sequence was as follows: Ala-Ser-Tyr-Thr-Val-Lys-Leu-Ile-Thr-Pro-Asp-Gly-Glu-Asn-Ser-Ile-Glu-Cys-Ser-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Ile-Thr-Ala-Gly-Ser-Val-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Ile-Glu-Ala-Gly-Tyr-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Glu-Thr-His-Lys-Glu-Glu-Asp-Leu-Tyr.
In an alignment of various ferredoxins with high homology from unicellular and filamentous blue-green algae, Synechocystis 6714 ferredoxin showed 4 gaps. Those between residues 9 and 10 and between residues 12 and 13 were unique for the ferredoxins from the unicellular algae Synechocystis 6714 and Aphanothece sacrum (ferredoxin I). Therefore, ferredoxins from unicellular algae were distinguishable from those of filamentous algae in terms of the presence of gaps. This feature appears to coincide with the phylogenetic division between the two types of blue-green algae.View full abstractDownload PDF (444K) -
Yasuhiro TAKAHASHI, Toshiharu HASE, Hiroshi MATSUBARA, Georgina N. HUT ...1982 Volume 92 Issue 5 Pages 1363-1368
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of Chlorogloeopsis fritschii ferredoxin was determined for its carboxymethylated derivative by using solid-phase sequencing, fragmentation with various enzymes, and manual Edman degradation procedures. The ferredoxin was composed of 98 amino acid residues and lacked methionine and tryptophan. The sequence was as follows: Ala-Thr-Tyr-Lys-Val-Thr-Leu-Ile-Asn-Asp-Ala-Glu-Gly-Leu-Asn-Gln-Thr-Ile-Glu-Val-Asp-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Ile-Lys-Ser-Gly-Thr-Val-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Ile-Glu-Ala-Gly-Tyr-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Glu-Thr-His-Lys-Glu-Glu-Glu-Leu-Tyr. A phylogenetic tree was constructed on the basis of a comparison of various algal ferredoxins and it was found that C. fritschii ferredoxin was closely related to Mastigocladus laminosus ferredoxin, though they are in different genera of the blue-green algae. Aspects of the taxonomy and molecular evolution of blue-green algal ferredoxins are discussed.View full abstractDownload PDF (483K) -
Kunio TSURUGI, Kikuo OGATA1982 Volume 92 Issue 5 Pages 1369-1381
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chromatin fraction of rat liver exhibited proteolytic activity caused by serine proteases, with maximal activity at pH 8 or 10. They were analyzed by affinity labeling with [3H]diisopropylfluorophosphate followed by SDS-polyacrylamide gel electrophoresis, and partially purified by gel filtration through Sepharose 6B after selective extraction from the chromatin fraction. The following results were obtained.
1. The chromatin fraction contained three DFP-binding proteins with molecular weights of 52, 000, 25, 000, and 15, 000 daltons, and they were tentatively designated proteins A, B, and C, respectively. Unlike proteins A and B, protein C reacted with DFP more strongly at pH 10 than at pH 8. A greater part of protein B was present in the nucleoli, while the others were predominantly distributed in extra-nucleolar chromatin.
2. Proteins A and B were extracted from the chromatin fraction by 5M urea and 0.7M NaCI, respectively; while protein C was not extractable by either solution. Proteins A and B were further purified by gel filtration through Sepharose 6B.
3. Protein B was a neutral protease with a maximal activity for 3H-labeled ribosomal proteins at pH 8 and showed high specificity for basic proteins, such as histone and ribosomal proteins. Protein A was an alkaline protease with a maximal activity at pH 10 and showed proteolytic activity not only for basic proteins but also for hydrophobic proteins, such as casein and non-histone proteins of chromatin.
4. Protein A was activated at pH 8 by high concentrations of NaCl, suggesting the presence of some inhibitor (s). Protein A was converted to protein C at pH 10, and also at pH 8 with high concentrations of NaCl. Thus, protein A is suggested to be the complex of protein C and unknown inhibitor (s).
5. When the chromatin fraction was incubated at pH 10, non-histone proteins were degraded much faster than at pH 8, although H1 histone was degraded at similar rates at both pHs.
These results indicate that the chromatin fraction of rat liver contains at least two kinds of serine proteases, B and C, and that protease B is probably involved in the metabolism of basic protein, especially H1 histone. Protease C, the greater part of which associates with some inhibitors to form protein A, may play its main role in the metabolism of non-histone proteins.View full abstractDownload PDF (1725K) -
Ichiro MATSUOKA, Keiko TAKEDA, Masamitsu FUTAI, Yuji TONOMURA1982 Volume 92 Issue 5 Pages 1383-1398
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe performed kinetic studies on the reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP (DNS-ATP), with E. coli F1-ATPase (EF1) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained.
1. One mol of EF1which contains nonexchangeable 2 mol ATP and 0.5 mol ADP, binds 3 mol of DNS-ATP. The apparent dissociation constant, in the presence of Mg2+, was 0.23 μm. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t1/2 of 35 s, and reached 3.5 times the original fluorescence level. Following the fluorescence increase, DNS-ATP was hydrolyzed, and the fluorescence intensity maintained its enhanced level.
2. The addition of an excess of ATP over the EF1-DNS-nucleotide complex, in the presence of Me2+, decreased the fluorescence intensity rapidly, indicating the acceleration of DNS-nucleotide release from EF1. ADP and GTP also decreased the fluorescence intensity.
3. DCCD markedly inhibited the accelerating effect of ATP on DNS-nucleotide release from EF1 and the EF1-DNS-ATPase or -ATPase activity in a steady state. On the other hand, DCCD only slightly inhibited the fluorescence increase of DNS-ATP, due to its binding to EF1and the rate of single cleavage of 1 mol of DNS-ATP per mol of α subunit of EF1.
4. In the presence of Mg2+, 0.65-0.82 mol of DNS-ATP binds to 1 mol of the isolated a subunit of EF1 with an apparent dissociation constant of 0.06-0.07 μm. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t1/2<1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated α subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated β subunit. DNS-ATP was also unhydrolyzed by the isolated β subunit.
5. EF1-ATPase was reconstituted from α, β, and γ subunits in the presence of Me2+ and ATP. The kinetic properties of the fluorescence change of DNS-ATP in the reaction with the reconstituted EF1-ATPase were quite similar to those of native EF1.
Most of our findings are consistent with a simple mechanism that the high affinity catalytic site and low affinity regulatory site exist in the α subunit and β subunit, respectively. However, the findings mentioned in (4) suggest that the binding of the α and β subunit, which is mediated by the γ subunit, induces conformational change (s) in the ATP binding site located probably in the a subunit, and that the conformational change (s) is essential to exert the full hydrolyzing activity.View full abstractDownload PDF (1248K) -
Mayumi SHIBUYA, Kazue NAGATA, Mitsuko OKADA1982 Volume 92 Issue 5 Pages 1399-1402
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enzyme and antigen activities of aspartate aminotransferase [EC 2. 6. 1. 1] isozymes in several tissues of rats fed on pyridoxine-deficient or control diet for 4 weeks were determined. The enzyme activities in liver (supernate, mitochondria), heart (supernate), brain (mitochondria), and muscle (mitochondria) preparations of the deficient rats were abnormally low, whereas those in other tissue preparations were similar to those of control rats. In liver and heart preparations, the antigen activities of the deficient rats were similar to those in controls, but in brain and muscle mitochondria they were abnormally low. These findings suggest that the activity and the amount of this enzyme are regulated in different ways in different tissues, and suggest that the coenzyme level affects metabolic regulation of the enzyme molecule.View full abstractDownload PDF (302K) -
Takayoshi WAKAGI, Takahisa OHTA1982 Volume 92 Issue 5 Pages 1403-1412
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBeef liver mitochondrial F1ATPase was inactivated by the 2', 3'-dialdehyde derivative of ethenoATP (εATP) in a pseudo-first order reaction. The kinetics of protection of the enzyme against inactivation by various nucleoside triphosphates (NTPs) revealed that the dial-εATP was bound to the catalytic site as an affinity label. Certain anions (sulfate or bicarbonate) were ineffective for protection. In the early phase of the reaction, inactivation was due to the binding of 1 mol dial-εATP per mol enzyme. In this phase, dial-εATP bound exclusively to the subunit β of the enzyme, indicating that the catalytic site is in this subunit. The fluorescence of the ethenoadenosine moiety, bound exclusively to the subunit β of the enzyme, was measured as a conformational probe of the catalytic site region. Addition of ATP or CTP to the labeled enzyme resulted in a decrease in the fluorescence intensity. GTP and other NTPs were less effective than ATP or CTP. The anions (sulfate of bicarbonate) suppressed the ability of ATP to decrease the fluorescence in a competitive manner. Quantitative analysis of these fluorescence changes suggested that they might originate from the binding of the NTP to the regulatory site of the enzyme. These findings are in good agreement with the two-site model proposed by us (Wakagi, T. & Ohta, T. (1981) J. Biochem. 89, 1205) which was deduced from the steady state kinetics of the NTPase reactions catalyzed by the F1ATPase.View full abstractDownload PDF (1519K) -
Shinji YOSHITAKE, Masayoshi IMAGAWA, Eiji ISHIKAWA, Yoshiro NIITSU, Ic ...1982 Volume 92 Issue 5 Pages 1413-1424
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mild and efficient procedure for conjugating rabbit Fab' and horseradish peroxidase using a maleimide compound was developed. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl) maleimide to introduce maleimide groups. Then, the maleimide-enzyme was allowed to react with thiol groups of Fab', and the conjugate formed was separated from unreacted components by gel filtration with Ultrogel AcA 44. In the peak fraction of the separated conjugate, 98% of peroxidase was associated with Fab' and 90% of antibodies was associated with peroxidase. The recoveries in the conjugate of peroxidase and Fab' incubated for conjugation were 65-74%. The conjugate formed appeared to be largely monomeric. Both the enzyme activity and antigen-binding activity of Fab' were fairly well preserved in the conjugate. The cross-link formed was stable at 4°C at least 6 months. Use of the conjugates obtained by this method gave greater sensitivity in sandwich enzyme immunoassay for human ferritin and human thyroid-stimulating hormone than conjugates prepared by the periodate method. The conjugation using N-hydroxysuccinimide ester of m-maleimidobenzoic acid provided a similar monomeric preparation but was less efficient.View full abstractDownload PDF (967K) -
Ikuo ASHIKAWA, Yoshifumi NISHIMURA, Masamichi TSUBOI, Mitsuo ZAMA1982 Volume 92 Issue 5 Pages 1425-1430
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA sample of fluorescently-labeled nucleosome core particles was prepared by combining N-(3-pyrene)-maleimide to each of the two cysteine residues of the two H3 histones in the chicken erythrocyte nucleosome core particle, by a method previously reported by Zama et al. (Nucl. Acids Res. 5, 3881 (1978)). The fluorescence spectra and fluorescence lifetime of the sample in solvents with various Na+ concentrations have been examined. Three different types of micro-environment of the fluorescent probe have been found: (i) emits a pyrene dimer fluorescence at 460 nm, (ii) emits a pyrene monomer fluorescence with a long (75-65 ns) lifetime, and (iii) emits a pyrene monomer fluorescence with a short (5-15 ns) lifetime. The manner of dislocation of the two H3 subunits from each other in each of the solutions with different salt concentrations was discussed.View full abstractDownload PDF (466K) -
Toshio OTSUKI, Masakata MORI, Masamiti TATIBANA1982 Volume 92 Issue 5 Pages 1431-1437
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCarbamoyl-phosphate synthetase II of higher animals, the first enzyme of de novo pyrimidine biosynthesis, forms a multienzyme complex with aspartate carbamoyl-transferase and dihydroorotase, the second and third enzymes of the pathway. The hypothesis that the complex serves to channel carbamoyl-phosphate, synthesized by the first enzyme of the complex, to the second enzyme was tested using a highly purified complex preparation from Yoshida ascites hepatoma cells (AH 13). Experimentally, aspartate carbamoyltransferase in the complex was allowed to compete with exogenously added ornithine carbamoyItransferase, another carbamoyl-phos-phate-utilizing enzyme, for carbamoyl-phosphate which was either synthesized endogenously or added exogenously. The ratios of amounts of the two enzymic products, carbamoyl-aspartate and citrulline, were compared. In the absence of enzyme stabilizers dimethyl sulfoxide or glycerol, a slight channeling of the inter-mediate in the complex was observed. The further addition of 5-phosphoribosyl 1-pyrophosphate, MgUTP (positive and negative allosteric effectors of carbamoyl-phosphate synthetase II), 30% (v/v) dimethyl sulfoxide or 30% (w/v) glycerol did not affect the extent of channeling. It was slightly increased in the presence of 7.5% (v/v) dimethyl sulfoxide plus 2.5% (w/v) glycerol. Any shift of the assay temperature, pH or concentration of MgATP or of the enzyme complex resulted in little further increase in the extent of channeling. Even when a larger amount of the enzyme complex was used to approximate physiological conditions, there was no increase in the extent of channeling either without or with allosteric effectors. MgUTP even abolished channeling under these conditions. These results indicate that carbamoyl-phosphate can be channeled in the multienzyme complex of AH 13 cells, but the extent of channeling is very small, contrary to expectation.View full abstractDownload PDF (533K) -
Issei MABUCHI1982 Volume 92 Issue 5 Pages 1439-1447
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of myosin, heavy meromyosin (HMM), α-actinin, tropomyosin, and calmodulin on the interaction between an actin-depolymerizing protein (depactin) from starfish oocytes and rabbit skeletal actin were investigated.
1. Alpha-actinin or tropomyosin did not affect the inhibitory effect of depactin on salt-induced polymerization of actin and did not induce actin polymerization when added to a mixture of actin and depactin in a buffer solution which was designed to keep actin in F-form. Myosin or HMM allowed polymerization of actin under similar conditions except that ATP was not included. Addition of ATP, inorganic pyrophosphate, adenyl-5'-yl imidodiphosphate, or ADP abolished the effect of myosin.
2. None of these proteins could keep actin in a polymerized form when depactin was added to the mixture of actin and these proteins in the presence of ATP. In the absence of ATP, however, myosin protected actin against depactin.
3. The association constant between actin and depactin was estimated from competitive binding experiments using HMM to be around 4.5×106 M-1 assuming the association constant between actin and HMM to be 3×109 M-1 (Greene, L. E. & Eisenberg, E. (1980) J. Biol. Chem. 255, 549-555).
4. Actin did not activate Mg-ATPase activity of HMM in the presence of depactin. From the above results, the mode of interaction of myosin with actin in the presence of depactin and the possible role of myosin in actin assembly in the cell are discussed.View full abstractDownload PDF (1997K) -
Kyoko KIKUCHI-TORII, Sueko HAYASHI, Hideko NAKAMOTO, Satoshi NAKAMURA1982 Volume 92 Issue 5 Pages 1449-1456
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCatalase from Aspergillus niger was purified to homogeneity as judged from the results of ultracentrifugation and polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 385, 000 as estimated from sedimentation measurements. Carbohydrate analyses showed that the catalase was a glycoprotein containing about 8.3% neutral sugar and 1.9% glucosamine. Under denaturing conditions, polyacrylamide gel electrophoresis revealed only one band with a molecular weight of 97, 000 daltons in gels stained for either protein or sugar, suggesting that the native enzyme consists of four subunits with covalently bound carbohydrate. In the reaction with inhibitors, A. niger catalase showed lower affinity than the “standard” catalases. The pK values for HCN, HN3, and HF were estimated to be 3.4 (at pH 7.4), 2.3, and 1.5 (at pH 4.2), respectively. In addition, the fungal enzyme reacts with methyl hydrogen peroxide in a very unusual way. Even after the addition of a large excess of the peroxide, only catalase compound I was formed, and compound II did not appear. Using this unique property of A. niger catalase, we obtained CD and MCD spectra of compound I uncontaminated by compound II. The magnitude of the positive CD peak of compound I in the Soret region was about half that of the native enzyme. The MCD spectrum obtained was better resolved than that of bovine liver catalase compound I in the visible region.View full abstractDownload PDF (1208K) -
Takeshi ENDO, Tomoh MASAKI1982 Volume 92 Issue 5 Pages 1457-1468
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with α-actinins from chicken striated muscles, or fast-skeletal muscle, slowskeletal muscle, and cardiac muscle. The gizzard α-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100, 000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle α-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle α-actinin and striated-muscle α-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard α-actinin was immunologically distinguished from striated-muscle α-actinins.
Gizzard α-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle α-actinin could. Temperature-dependent competition between gizzard α-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard α-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard actoskeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle α-actinin molecules, they function similarly in vitro, and that gizzard α-actinin can interact not only with smoothmuscle actin (γ-and β-actin) but also with skeletal-muscle actin (α-actin).View full abstractDownload PDF (4202K) -
Masateru NISHIHARA, Hiroyuki MORII, Yosuke KOGA1982 Volume 92 Issue 5 Pages 1469-1479
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBis (monoacylglycero) phosphate (BMP) was found in an alkalophilic bacterium, Bacillus sp. A-007. This lipid comprised 4% of the phospholipid of this organism. The very low rate and extent of triphenylmethylation of BMP indicated that the two fatty acid residues occupied the C-1 and C-3' positions of the two glycerol moieties. The stereochemical configuration of the backbone of BMP was identified as sn-glycero-3-phosphory1-1'-sn-glycerol by sn-glycerol 3-phosphate dehydrogenase analysis after acetic acid hydrolysis and mild alkaline methanolysis. This configuration was the same as that of phosphatidylglycerol, but differed from that of BMP from mammalian cells. Finally, the structure of BMP in Bacillus sp. A-007 was concluded to be 1-acyl-sn-glycero-3-phosphory1-1' (3'-acy1)-sn-glycerol.
The fatty acids of BMP were those common in Bacillus species. The fatty acid composition of BMP was very similar to that of the major lipids in this organism. The fatty acid distribution between the C-1 and C-3' positions of BMP was determined by a newly developed method which included successive steps of acetic acid hydrolysis, chemical acylation and phospholipase A2 treatment. Fatty acids with longer chain length were concentrated in the C-1 position, and the shorter fatty acids were the major ones esterified at the C-3' position.View full abstractDownload PDF (1436K) -
Chiaki ROKUKAWA, Yasunori KUSHI, Kunihiro UENO, Shizuo HANDA1982 Volume 92 Issue 5 Pages 1481-1488
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGangliosides were isolated from rat liver and erythrocytes by chromatography on columns of DEAE-Sephadex and Iatrobeads, and finally purified by preparative TLC. The chemical structures of the purified components were studied by carbohydrate analysis, methylation analysis, sialidase treatment, fatty acid analysis and direct mass spectrometry. In rat liver, gangliosides GM3, GM1, GD3, GD1a, GD1b, and GT1b were identified. Gangliosides in rat erythrocytes were characterized as GM1, fucosyl-GM1, and GD1a. Sialic acid was the N-acetyl type only and lignoceric acid was the main fatty acid in all components of rat liver and erythrocytes.View full abstractDownload PDF (1239K) -
Koichi HIRAGA, Goro KIKUCHI1982 Volume 92 Issue 5 Pages 1489-1498
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycine decarboxylase, tentatively called P-protein, was inactivated when it was incubated with glycine in the presence of the aminomethyl carrier protein, called H-protein. The inactivation was accompanied by a spectral change in the P-protein as a pyridoxal phosphate enzyme; the spectrum became unusual, with a peak at 330 nm. The fluorescence emission spectrum of the inactivated P-protein showed a distinct peak at 390 nm when excited at 325 nm. Such a spectral change and concomitant inactivation of the P-protein could be completely prevented by the addition of sodium bicarbonate, which initiates the glycine-CO2 exchange in the reaction mixture. The inactivated P-protein was associated with H-protein and the methylene carbon of glycine, but not the carboxyl carbon, in a manner not separable by gel filtration, although the molar ratios of those three components were not constant. The H-protein recovered in the inactivated P-protein fraction was also catalytically inactive. Neither the pyridoxal derivative nor the methylene carbon of glycine appeared to be covalently bound with the protein, and the methylene carbon could be recovered as formaldemethone when treated with dimedone.
The inactivation of the P-protein appears to represent a suicide reaction of the P-protein as a side reaction of the glycine decarboxylation, which is supposed to involve the formation of a ternary complex of P-protein, aminomethyl moiety of glycine and H-protein through a Schiff base linkage of the H-protein-bound aminomethyl moiety with the pyridoxal phosphate of P-protein.View full abstractDownload PDF (1552K) -
Yoko KITA, Shinji SAKAGUCHI, Yasunori NITTA, Takehiko WATANABE1982 Volume 92 Issue 5 Pages 1499-1504
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe modification of Taka-amylase A (TAA) [EC 3. 2. 1. 1] of Aspergillus oryzae by diethylpyrocarbonate (DEP) was carried out at 25°C and at pH 5.8 (0.1M acetate buffer). Two out of the six histidine residues were modified with 4.6mM DEP, and two or three histidine residues were modified with 23mM DEP. In both cases, one of them was protected from modification by the presence of 15% maltose. The results suggest that two or three out of the six histidine residues are exposed on the surface of the TAA molecule, and one of them exists near the maltose binding site.
Ethoxycarbonylation of histidine residues of TAA caused loss of the amylase activity and activation of the hydrolysis of phenyl α-maltoside (øαM). The kinetic parameters of the modified TAA for several substrates and analogs were determined at 25°C and at pH 5.3 (0.08M acetate buffer). From the results, it was found that this alteration of the enzyme activity by the modification was not due to a change in Km value but to a change in ko value. Thus, some of the histidine residues in TAA are suggested to play an important role in the enzyme catalytic function.View full abstractDownload PDF (426K) -
Kazuhiro YAMADA, Kaoru KOMETANI1982 Volume 92 Issue 5 Pages 1505-1517
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have used the method of enthalpy titration to analyze the structural changes of troponin C caused by calcium binding. Successive additions of calcium to metalfree troponin C in a microcalorimeter cell result in at least three distinguishable transitions. Analysis of the results has shown that troponin C has at least three classes of calcium binding sites; one site of highest affinity (binding constant, 108-1010 M-1), one site of next highest affinity (binding constant, 106-107 M-1) and two low affinity sites (binding constant, 105-106 M-1). Titrations of troponin C with calcium at various temperatures have shown that calcium binding causes large changes in the heat capacity of troponin C. Following the method of Sturtevant (1977), the magnitudes of the hydrophobic and intramolecular vibrational contributions to the heat capacity and entropy changes of troponin C on calcium binding have been estimated. In Mg-free solutions, calcium binding to the 1st site of highest affinity gives rise to a strong hydrophobic effect and to a tightening of the molecular structure. In contrast, calcium binding to the 2nd site of next highest affinity gives rise to a strong hydrophobic effect in the reverse direction and to a “softening” of the structure. Calcium binding to the two low affinity sites has a moderately strong hydrophobic effect and also causes a moderate tightening of the structure. These results are in many respects similar to those obtained with proton magnetic resonance spectroscopy by Levine et al. (1977). These studies are mutually complementary. When 1 mM magnesium is present the changes caused by calcium binding to the two high affinity sites are greatly altered, whereas those involving the two low affinity sites are not much affected. The moderate tightening of the structure which is caused by calcium binding to the two low affinity sites, and which is seen both in the absence and presence of magnesium, is most likely to be involved in the regulation of contraction.View full abstractDownload PDF (986K) -
Keiichi YAMAMOTO, Takamitsu SEKINE1982 Volume 92 Issue 5 Pages 1519-1525
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of N6-[(6-aminohexyl) carbamoylmethyl] ATP and its dinitrophenyl derivative as substrates of myosin were studied to test whether or not these ATP analogues are suitable for the affinity chromatography of myosin. The Km and Vmax values of heavy meromyosin for ATP, N6-[(6-aminohexyl)carbamoylmethyl]-ATP, and its dinitrophenyl derivative were 11.9 μM and 0.492 μmol P1/min•mg, 137 μm and 0.454 μmol P1min•mg, and 250 μm and 3.96 μmol P1/min•mg, respectively in 0.5M KCI, 5mM CaCl2, 20mM Tris-rnaleate buffer, pH 7.0 at 250°C. The association rate constant of N6-[6-aminohexyl) carbamoylmethyl] ATP to the active site of myosin was only 8% of that of ATP. The reason seemed to be the electrostatic repulsion between the positively charged group in the active site and the amino group of N6-[(6-aminohexyl) carbamoylmethyl]ATP because the rate constant of the dinitrophenyl derivative was 38% of that of ATP. It is suggested, therefore, that when this ATP analogue is attached to a solid matrix through its amino group, the association rate constant could be improved.
It was shown that the dinitrophenyl derivative of N6-[(6-aminohexyl) carbamoyl-methyl]ATP was trapped in the active site of heavy meromyosin by the cross-linking of SH1 and SH2 with p-phenylenedimaleimide. However, the stability of the dinitrophenyl derivative in the active site was very low compared to that of ATP. These results suggested that this ATP analogue interacts with myosin through the active site and that, if appropriate conditions to stabilize the complex with myosin are found, a column prepared with this analogue will be a useful tool for the affinity chromatography of myosin.
We suggest that the mechanism of nucleotide trapping is a conformational change in the active site which slows down the rate of product release rather than a steric blocking of the active site by the cross-linking reagent.View full abstractDownload PDF (534K) -
Ikuko TAKAHASHI, Kyozo OGURA1982 Volume 92 Issue 5 Pages 1527-1537
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUndecaprenyl pyrophosphate synthetase and geranylgeranyl pyrophosphate synthetase were partially purified and characterized from Bacillus subtilis, from which heptaprenyl pyrophosphate synthetase and farnesyl pyrophosphate synthetase had previously been obtained [Takahashi et al. (1980) J. Biol. Chem. 255, 4539; (1981) J. Biochem. 89, 1581]. The undecaprenyl pyrophosphate synthetase catalyzed the Z-oligomerization of isopentenyl units with farnesyl pyrophosphate as a priming substrate to give C50 and C55 prenyl pyrophosphates with Z, E mixed stereochemistry. Various geometric isomers of C10, C15, C20, and C25 prenyl pyrophosphates also acted as priming substrates to give the corresponding isomeric products with chain lengths of C50 and C55, including unnatural products. In addition to absolute requirements for Mg2+ and detergent, the enzyme activity was further stimulated markedly by monovalent cations such as K+ and NH4+. The geranylgeranyl pyrophosphate synthetase catalyzed C5→C10→C15→C20 reactions to give E, E-farnesyl and E, E, E-geranylgeranyl pyrophosphates. This enzyme was not affected by monovalent cations or detergent.View full abstractDownload PDF (812K) -
Shohei MIYATA, Hirozi K. KIHARA1982 Volume 92 Issue 5 Pages 1539-1546
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA DNA-binding protein, which migrated as one major protein band, with a molecular weight of 14, 000, on sodium dodecylsulfate polyacrylamide gel, was purified from a culture medium of mouse thymus cells. The interaction of the isolated protein with DNA in vitro was assayed by a nitrocellulose filter binding technique. Equilibrium competition experiments demonstrated that the DNA-binding protein had the ability to differentiate among sequences of polynucleotides, indicating that the DNA-binding protein-DNA interaction was at least partially specific. This protein increased the helix melting temperature of DNA and inhibited the incorporation of [3H]dTMP into DNA by the DNA polymerase of calf thymus in vitro.View full abstractDownload PDF (598K) -
Shigeo SERIZAWA, Hideaki OTSUKA, Yousuke SEYAMA, Tamio YAMAKAWA1982 Volume 92 Issue 5 Pages 1547-1557
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence of cholestanol had never been reported in cultured cells, but in the present study cholestanol was detected both in human and rat skin fibroblasts. The present study has shown that the origin of cholestanol in these fibroblasts was derived from de novo synthesis in the cells rather than from the fetal calf serum used in the growth medium. The activity of 5a-cholestan-3-one reductase which is the enzyme catalyzing the final step in the biosynthesis of cholestanol was measured by mass fragmentography using deuterium labeled 5a-cholestan-3-one as a substrate. The activity of this enzyme was also detected in the microsomal fractions of rat cerebrum and cerebellum as well as in the liver. These results suggest that cholestanol can probably be synthesized in extra-hepatic tissues as well as in the liver.View full abstractDownload PDF (864K) -
Takashi KUMAZAKI, Shin-ichi ISHII1982 Volume 92 Issue 5 Pages 1559-1566
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive R-type pyocins have been reported which are almost identical with one another in their morphology and subunit composition, though distinct in receptor-binding specificity. We isolated fibers from pyocin R2, R3, and R4 by essentially the same procedure as used in our previous isolation of pyocin R1 fiber with unimpaired receptor-binding ability. All the isolated fibers including RI fiber were indistinguishable from one another, in terms of electron microscopic observation and subunit composition analysis by SDS gel electrophoresis. They consisted mainly of Subunit No. 2 (Mw 71, 000) and No. 9 (31, 000) proteins. Although Subunit No. 9 protein in every fiber was susceptible to trypsin and afforded a fragment with the same molecular weight (about 19, 000) detectable in the SDS gel, Subunit No. 2 protein was cleaved with trypsin only after the fiber had been treated with an organomercurial, 4-(p-sulfophenylazo)-2-mercuriphenol. The cleavage of Subunit No. 2 protein proceeded to give several fragments with molecular weights ranging from 64, 000 to 34, 000, and the fragmentation patterns were electrophoretically distinct at least among R1 fiber, R3 fiber, and others (R2 and R4 fibers). The results indicate that Subunit No. 2 proteins of these fibers are different from one another in the structure surrounding trypsin-susceptible peptide bonds. Immunological investigations with anti-R1 fiber antibodies provided some additional information on the difference among R-type pyocins at the fiber level.View full abstractDownload PDF (2088K) -
Shin-ichi NIHIRA, Jiro KOYAMA1982 Volume 92 Issue 5 Pages 1567-1576
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inhibitory effects of diisopropyl fluorophosphate (DFP) and p-tosyl-L-lysine chlorornethyl ketone (TLCK) on the ingestion and digestion of 125I-α-amylase [BαA, EC 3. 2. 1. 1] complexed with antibody by guinea pig peritoneal macrophages were studied by sucrose density gradient fractionation. When macrophages were exposed to the complex at 37°C and homogenized after washing, most of the trichloroacetic acid (TCA)-insoluble radioactivity was recovered in two fractions which behaved like the plasma membrane and lysosomes in the density gradient, and the TCA-soluble radioactivity was found in the fraction not moving into the gradient. The complex bound to Fc receptors on the cell surface, therefore, was shown to be internalized, transported to the lysosomes, and finally digested to amino acid catabolites.
The inhibition with DFP caused an accumulation of the complex in a region between the plasma membrane and lysosomes. This distribution of radioactivity resembled that of cytochalasin B-inhibited macrophages. TLCK also inhibited the digestion of the complex, but the distribution of radioactivity revealed a marked accumulation of the complex in the region of lysosomes. These results provide strong support to the hypothesis that DFP blocks the ingestion of the complex, as in the case of cytochalasin B, whereas TLCK inhibits the intralysosomal proteolysis of the complex.View full abstractDownload PDF (858K) -
Hisao FUJISAKI, Hiroshi ASAI1982 Volume 92 Issue 5 Pages 1577-1583
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEnzymatic characteristics of heavy meromyosin (HMM) were investigated by measuring β-naphthyl triphosphate (β-NapP3) hydrolysis in the presence and absence of F-actin. β-NapP3 hydrolysis by HMM was inactivated by F-actin in the presence of Mg ions; in the presence of sufficient F-actin, the activity was about one-half of that in the absence of F-actin. In the presence of Ca ions the activity disappeared almost completely on addition of sufficient F-actin. Two different values of the Michaelis constant (Km) were obtained from the data for β-NapP3 hydrolysis by HMM in the presence of Mg ions; one of them vanished in the presence of sufficient F-actin, and only one Km value was obtained in the presence of Ca ions. These results suggest the existence of two distinct enzymatic sites in HMM, one inactivated by F-actin in the presence of Mg or Ca ions, the other inactive in the presence of Ca ions but active in the presence of Mg ions and not influenced by F-actin. Use of subfragment-1 (A1) (S-1 (A1)) and S-1 (A2) instead of HMM confirmed that these enzymatic characteristics are not due to a difference in the alkali light chains on myosin heads.View full abstractDownload PDF (453K) -
Hidenori HAYASHI, Masataka SHITARA, Fumie YAMASAKI1982 Volume 92 Issue 5 Pages 1585-1590
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe origin of the lipids accumulated in liver lysosomes after administration of Triton WR-1339 was investigated. When Triton WR-1339 was injected into rats, serum triglyceride and cholesterol increased markedly. The highest content of triglyceride was observed in the second-day serum, from which very-low-density lipoprotein (VLDL) was isolated. The VLDL was administered to normal rats, then the light mitochondrial fraction of the liver at 24 h was centrifuged in a sucrose density gradient. The activities of lysosomal enzymes, acid phosphatase, N-acetyl-β-D-glucosaminidase and acid lipase, were all shifted to less dense fractions as compared with those of normal lysosomes. [3H]Triglyceride-labeled VLDL was injected similarly, and at 12 and 24 h after the administration, the light mitochondrial fraction of the liver was fractionated by sucrose gradient centrifugation. Protein content and radioactivity in the immunoprecipitate with anti-VLDL serum at 12 h showed almost the same distribution as acid phosphatase activity. At 24 h, though acid phosphatase activity, immunoprecipitable protein content and radioactivity were all found in less dense fractions than in the case of normal lysosomes, the former two distributions were significantly different from the latter. The anti-VLDL serum reacted in Ouchterlony tests not only with Triton-induced VLDL and normal VLDL but also with the extract from low-density lysosomes.
These results suggest that the lipids accumulated in low-density lysosomes following the administration of Triton WR-1339 were probably derived from the elevated serum VLDL induced by the treatment.View full abstractDownload PDF (446K) -
Kazuyuki SASAKI, Atsunori KASHIWAGI, Kiichi IMAMURA, Takehiko TANAKA1982 Volume 92 Issue 5 Pages 1591-1598
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of ornithine decarboxylase-inducing factor, which was partially purified from ascites fluid of mice bearing Ehrlich ascites tumor, on ornithine decarboxylase activity in normal mouse liver was studied. The enzyme activity started to increase 18 h following intraperitoneal single injection of the factor, reached a maximum at 20 h and then declined gradually. Actinomycin D administered at 16 or 18 h after the factor treatment caused a further increase in the activity of ornithine decarboxylase, so-called superinduction, though the enzyme activity was still very low at each time point. On the other hand, other inhibitors of RNA synthesis, cordycepin and α-amanitin, did not affect the enzyme activity at each time point. This finding casts considerable doubt on the Tomkins model for the superinduction, as already pointed out by several investigators. Injection of actinomycin D after the factor-mediated increase in enzyme activity had reached a maximum maintained the increased level for at east 4 h. In contrast, the factor-mediated enzyme induction and superinduction were completely prevented if the drug was given at 14 h. In addition, actinomycin D injected within 14 h following the administration of the factor did not exert any effect on the factor-mediated enzyme induction. Cycloheximide and 1, 3-diaminopropane inhibited both the factor-mediated enzyme induction and the superinduction by actinomycin D. When the liver extract obtained from the animals treated with the factor alone was mixed with that from the factor-injected animals treated with 1, 3-diaminopropane at a volume ratio of one to one, and also when the liver extract from the factor-injected animals treated with actinomycin D was mixed with that from the factor-injected animals treated with diaminopropane in the same volume ratio, the enzyme activities were reduced to 73 and 71% of the expected levels, respectively. However, when the liver extract from the animals treated with factor alone was mixed with that from the factor-injected animals treated with actinomycin D, the activity showed an additive effect. Furthermore, the apparent half-life of ornithine decarboxylase was not influenced by treatment of actinomycin D (81 and 82 min with and without the drug treatment, respectively).
The results of the experiments described above suggest that the factor-mediated ornithine decarboxylase induction was due to de novo synthesis of the enzyme following new mRNA synthesis which occurred at a very limited period between 14 and 16 h after the factor injection. In addition, the presence of mRNA for the enzyme was necessary for the superinduction.View full abstractDownload PDF (613K) -
Kunio FUJIWARA, Akiko OGAWA, Hisatsugu ASADA, Hitoshi SAIKUSA, Hideki ...1982 Volume 92 Issue 5 Pages 1599-1605
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU to mercaptosuccinylated bovine serum albumin via a cross-linker, N-(γ-maleimidobutyryloxy) succinimide. Enzyme labeling of PU was performed using β-D-galactosidase [EC 3. 2. 1. 23] via N-(m-maleimidobenzoyloxy) succinimide. An ultrasensitive and specific enzyme immunoassay for PU was developed utilizing these reagents by a double antibody technique. The standard curve of the assay was linear in the range of 2 pg to 100 pg, and the lower limit of detection was 28.2 pΜ (2 pg/tube); so the enzyme immunoassay was found to be approximately 326, 000 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is free from interference by any purine or pyrimidine analogs, or by other drugs commonly used for the inhibition of protein synthesis. Using this assay, drug levels were easily determined in rat tissue following PU administration. Since PU is extensively available as an inhibitor of protein synthesis, the enzyme immunoassay should provide useful tool for developing biochemical and toxicity studies of PU.View full abstractDownload PDF (535K) -
Kazunobu MATSUSHITA, Emiko SHINAGAWA, Osao ADACHI, Minoru AMEYAMA1982 Volume 92 Issue 5 Pages 1607-1613
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane-bound cytochromes c of Pseudomonas aeruginosa grown aerobically were investigated. By detecting polypeptides with heme-catalyzing peroxidase activity on a sodium dodecyl sulfate polyacrylamide gel, four major (Band I, 33, 000 daltons; II, 25, 000; III, 20, 000; IV, 16, 000) and one minor (V, 11, 500) hemoproteins were found in the membrane fraction, while one hemoprotein (VI, 8, 200) was detected in a small amount in the cytosol fraction. All these hemoproteins (bands I to VI) appeared to be cytochromes c, because all bands were detected even after being treated with HCl-acetone.
Of the membrane-bound cytochromes c, cytochromes c-551 (band IV) and c-555 (band V) were solubilized with Triton X-100 and purified by repeated DEAE-cellulose column chromatography. Both purified cytochromes c-551 and c-555 were monomeric and their molecular weights were estimated to be 16, 400 and 11, 500, respectively. Their respective midpoint potentials were 0.31 and 0.34 V, and their respective isoelectric points in the reduced form were 3.8 and 5.2.
The purified cytochromes c-551 and c-555 were found to be clearly different from “soluble” cytochrome c-551, and might function in the membrane-bound aerobic respiratory chain of P. aeruginosa.View full abstractDownload PDF (934K) -
Kiyohito SHIMURA, Ken-ichi KASAI1982 Volume 92 Issue 5 Pages 1615-1622
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe devised a new separation technique for the protein, “affinophoresis, ” which is based on its specific affinity and utilizes electrophoresis. This technique requires a carrier macromolecule, “affinophore, ” which contains both an affinity ligand for a certain protein and many charges, either positive or negative, in order to migrate rapidly in an electric field. When a mixture of proteins is electrophoresed in the presence of the affinophore, the protein having an affinity with the ligand will form a complex with the affinophore. This results in a change in the apparent electrophoretic mobility. If the protein is sufficiently accelerated, we can separate it from other materials.
A cationic affinophore for trypsin was prepared. Soluble dextran (MW_??_10, 000) was coupled with a DEAE-group and m-aminobenzamidine, a competitive inhibitor of trypsins. Electrophoresis of trypsins from several origins on agarose gel plates in the presence of the affinophore showed that affinophoresis actually occurred. The electrophoretic mobilities of trypsins increased towards the cathode, the same direction as the affinophore movement. The presence of leupeptin and treatment of the trypsins with TLCK suppressed the effect of the affinophore. Streptomyces griseus trypsin, contained in Pronase, was easily separated and detected.
This procedure is distinct from affinity chromatography and so-called affinity electrophoresis in that the support of the affinity ligand moves, and has advantages especially for analytical purposes: for example, the detection of specific molecules regardless of their isoelectric points.View full abstractDownload PDF (2619K) -
Tomoko TAKAHASHI, Yukiko TSUCHIDA, Masachika IRIE1982 Volume 92 Issue 5 Pages 1623-1633
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo inactive fragments of glucoamylase [EC 3. 2. 1. 3] from a Rhizopus sp. were isolated from the glucoamylase fraction obtained by CM-Sephadex chromatography in the previous purification of the glucoamylase (T. Takahashi et al. (1978) J. Biochem. 84, 1183-1194); the fraction contained at most 2.5% of the fragments, besides a mixture (97.5%) of three glucoamylases, designated as Glue, (M. W. 74, 000), Gluc2 (M. W. 58, 600), and Gluc3 (M. W. 61, 400) in order of content. The two isolated fragments, named fragments H and L in order of size, were found to be homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation analysis. The molecular weights of fragments H and L were 16, 700 and 14, 400, respectively. Fragment H had alanine as its N-terminal residue although fragment L was heterogeneous at the N-terminal amino acid. The N-terminal amino acids of Gluc1, Gluc2, and Gluc3 were found to be alanine, glutamic acid, and lysine, respectively; the three enzymes had the same C-terminal amino acid sequence of -Ser-Ala•OH. Immunodiffusion demonstrated that both fragments cross-reacted with Gluc1 and Gluc1 but not with Gluc2, although the three enzymes had common antigenicity. These results, together with the amino acid and sugar compositions of the fragments, indicate that the two fragments were derived from the N-terminal glycopeptide moiety of Gluc1 by the action of a proteolytic enzyme (s) with concomitant formation of Gluc2; Gluc3 also seems to be produced by proteolytic modification of Gluc1 with loss of its N-terminal peptide moiety.View full abstractDownload PDF (3260K) -
Kazuko WATANABE, Takashi KITAURA, Masahiro YAMAGUCHI1982 Volume 92 Issue 5 Pages 1635-1641
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Crayfish myosin B lost Ca2+-dependent regulation in superprecipitation upon addition of pure rabbit skeletal F-actin.
2. Actomyosin reconstituted from crayfish myosin and pure rabbit skeletal F-actin showed both Ca2+-dependent regulation in superprecipitation and Mg2+-ATPase activity upon addition of native tropomyosin prepared from crayfish or rabbit skeletal muscles. Also, superprecipitation of this actomyosin was induced by ITP without Ca2+-dependent regulation, as is the case in rabbit skeletal actomyosin.
3. Actomyosin reconstituted from crayfish native thin filament and crayfish or rabbit skeletal myosins showed Ca2+-dependent regulation.
4. These findings indicate that crayfish myosin is similar to rabbit skeletal myosin and different from chicken gizzard myosin in regulatory function.View full abstractDownload PDF (999K) -
Noriko SATOH, Morio SETAKA, Toshio HONGO, Takao KWAN, Hideo UTSUMI1982 Volume 92 Issue 5 Pages 1643-1647
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlatelets were found by a spin label technique to orient on flat surfaces of glass as well as of Teflon. This kind of platelet orientation was not caused by centrifugation or partial dehydration of the membrane preparation as employed usually to make oriented planar multilayers of biological membranes on the surface of a supporting plate (1-8), but was considered to be closely related to the adhesion or the aggregation properties of platelets. The amount of oriented platelets varied depending on the platelet treatment and was estimated from a computer simulation of the observed ESR spectra to be about one-half of that of the non-oriented ones in the case of thrombin-treated platelets. This technique may be useful as a new tool to explore the adhesion or aggregation properties of platelets.View full abstractDownload PDF (340K) -
Michinao MIZUGAKI, Tomoko NISHIMAKI, Takayuki SHIRAISHI, Akihiko KAWAG ...1982 Volume 92 Issue 5 Pages 1649-1654
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated. trans-2-[6, 7, 8-2H7] Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4 R- or 4 S-[4-2H1] NADPH. The deuteriumlabeled octenoyl-CoA was also incubated with the reductase in the presence of NADPH in 2H2O. Aliquots of octanoic acids formed were analyzed, after esterification, by gas chromatography-mass spectrometry (GC-MS) to demonstrate that the pro-4 R hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA and that hydrogen from water was introduced into the C-2 position of octenoyl-CoA.
The remaining portions of octanoic acids isolated from the incubation mixtures were converted to their CoA esters by the action of acyl-CoA synthetase, and they were dehydrogenated by treatment with acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2 R and pro-3 R hydrogen atoms of acyl-CoA. The deuterium contents of the products were also analyzed by GC-MS, and the results indicated that the reduction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase occurred by an anti-addition of hydrogen via a 2-Re, 3-Re attack on the trans-double bond of the substrate.View full abstractDownload PDF (416K) -
Jaap H. WATERBORG, Charles M. A. KUYPER1982 Volume 92 Issue 5 Pages 1655-1661
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe alkaline zinc-metallo nuclease of Physarum polycephalum is an endonuclease with a high specificity for single-stranded nucleic acids. Single-stranded DNA was cleaved at least 6, 000 times faster than double-stranded DNA under identical conditions. In the supercoil-induced single-stranded region of Form I PM 2 DNA only a single nick was made.
The nuclease showed nucleotide specificity. Poly (A), poly (I), and poly (dT) were preferentially hydrolyzed.
Product analysis showed that it acted by an endonucleolytic mechanism: long polynucleotides were fragmented via intermediate length products to oligo- and mono-nucleotides with the phosphate group at the 5'-terminal position.
Extensive similarities exist with the single-strand-specific nuclease S 1 from Aspergillus. The zinc-metallo endonuclease from Physarum could be used as a similar probe for single-stranded nucleic acids at neutral or alkaline pH conditions.View full abstractDownload PDF (989K) -
Kazuo TERAO, Toshio UCHIUMI, Kikuo OGATA1982 Volume 92 Issue 5 Pages 1663-1666
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen rat liver 80 S ribosomes or polysomes were dissociated into their subunits by EDTA-treatment, a 5 S RNA-L 5 protein complex was present in EDTA-derived small subunits, and released from the subunits by urea. The interaction of 5 S RNP with the small subunits did not occur in the case of 80 S couples formed from 40 S and 60 S subunits in the presence of Mg2+, indicating that the attachment of 5 S RNP to EDTA-derived small subunits was not an artifact caused by the unfolding of the subunits. These results give direct evidence that 5 S RNP is located at the interface between large and small subunits, and suggest that 5 S RNP binds to the 40 S subunit in the functioning polysomes, but not the 80 S couples.View full abstractDownload PDF (1606K) -
Mikio KATAOKA, Osamu HISATOMI, Fumio TOKUNAGA, Hiroshi WASHIOKA, Akira ...1982 Volume 92 Issue 5 Pages 1667-1670
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural changes during the thermal phase transition of purple membrane were observed by freeze-fracture electron microscopy. Native Halobacterium halobium cells contain broad purple membrane areas about 1 μm in diameter. The boundary separating purple and red membranes is obvious. On warming at 80°C, particles of red membrane spread out beyond the boundary. Then a purple membrane area is eventually divided into small areolae of similar size, about 0.1 μm in diameter. By cooling down slowly, the purple membrane area is reformed and the crystalline arrangement also restored.View full abstractDownload PDF (2154K) -
Michinao MIZUGAKI, Chiharu KIMURA, Tomoko NISHIMAKI, Hirotaka YAMAMOTO ...1982 Volume 92 Issue 5 Pages 1671-1674
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS2, 4-Dienoyl-CoA reductase has been separated from Escherichia coli grown in the presence of linoleic acid and purified to homogeneity. The enzyme has a molecular weight close to 50, 000 as determined by gel filtration on Sephacryl S-200 Superfine. The reductase was rather stable in a buffer containing citric acid and kept its full activity on heating at 55°C for 10min in the pH range of 5.5 to 6.5, but was completely inactivated on heating at 58°C for 10min. Phosphocellulose column chromatography revealed that the reductase was not involved in the multi-enzyme complex (molecular weight of 260, 000) of fatty acid oxidation.View full abstractDownload PDF (301K) -
Minoru NOMOTO, Yoshimasa KYOGOKU, Koichi IWAI1982 Volume 92 Issue 5 Pages 1675-1678
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous study showed that the amino acid sequence of Tetrahymena histone H 2 B has a blocked N-terminal residue followed by 119 amino acid residues, X-Pro-Lys…… [J. Biochem. (1982) 91, 897-904]. Now, X has been found to be N-trimethylalanine by 1H nuclear magnetic resonance spectroscopy of the isolated X-Pro-Lys and X. This identification was supported by mass spectrometry of X-Pro-Lys and by column and paper chromatography of X. This is the first time that N-terminal blocking by methylation has ever been found in histones.View full abstractDownload PDF (208K) -
Toshihiro KITAMOTO, Masae NAKASHIMA, Atsushi IKAI1982 Volume 92 Issue 5 Pages 1679-1682
Published: November 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHen egg white ovomacroglobulin purified by Miller and Feeney without reference to its activity was shown to have a protease inhibitory activity towards trypsin, papain, and thermolysin. It has four subunits of equal molecular weight (175, 000 by SDSPAGE) and each two of which are disulfide bonded. Upon incubation with trypsin it yields a fragment of Mr=80, 000 plus smaller ones. The subunit composition, amino acid composition and a newly found protease inhibitory activity place ovomacroglobulin as a closely related protein to human serum α2-macroglobulin.View full abstractDownload PDF (870K)
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