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Volume 89 (1981)
- Issue 6 Pages 1667-
- Issue 5 Pages 1347-
- Issue 4 Pages 979-
- Issue 3 Pages 701-
- Issue 2 Pages 337-
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Volume 89, Issue 2
Displaying 1-42 of 42 articles from this issue
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Akihiko KAWAGUCHI, Tsutomu YOSHIMURA, Shigenobu OKUDA1981 Volume 89 Issue 2 Pages 337-339
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method is described for the preparation of CoA thioesters of fatty acids. Acyl-CoA thioester was synthesized by way of 1-acylimidazole and purified by high performance liquid chromatography. This method is applicable to the preparation of a variety of acyl-CoA thioesters of various chain lengths (from C2 to C18) and with varying degrees of unsaturation. It is particularly suitable for preparing acyl-CoA thioesters of labeled fatty acids or fatty acids available only in small amounts.View full abstractDownload PDF (219K) -
Kazuhiro KOHAMA, Howard HOLTZER1981 Volume 89 Issue 2 Pages 341-349
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS“DNase binding protein” of 43K daltons as determined by SDS-polyacrylamide gel electrophoresis, was purified from the 0.1M KCl-soluble (non-structural) fraction of chicken skeletal muscle. The protein was distinct from actin in amino acid composition and physicochemical properties.
“DNase binding protein” was also isolated from other kinds of muscle and non-muscle cells. The ratio of the amino acid incorporation rate of “DNAase binding protein” to that of actin is different between skeletal and smooth muscle and nonmuscle cells.View full abstractDownload PDF (1205K) -
Yoshio IMAI1981 Volume 89 Issue 2 Pages 351-362
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe roles of cytochrome b5 in NADPH-dependent monooxygenase reactions catalyzed by reconstituted systems containing four forms of hepatic microsomal cytochrome P-450, i.e. P-4501, P-4502, P-4481, and P-4482, were examined. Various substrates were metabolized actively in the absence of cytochrome b5 by the system containing P-4501, but the monooxygenase reactions were accompanied by oxidation of NADPH uncoupled to the product formation. When cytochrome b5 was included in the system, the product formation increased to various extents, depending on the substrates used, while NADPH oxidation changed much less, resulting in an improvement of the coupling efficiency. The increase was large when a substrate metabolized at a low velocity was employed. Evidence is presented that the second of two electrons required for the monooxygenase reactions could be introduced into P-4501 via cytochrome b5. On the other hand, the rate of P-4501 reduction was not affected by the addition of cytochrome b5 to the system and that of cytochrome b5 reduction by NADPH-cytochrome P-450 reductase was sufficient to support electron flow to cytochrome P-450 via cytochrome b5 as the second electron. The stimulatory effect of cytochrome b5 on the P-4501-catalyzed monooxygenase reactions can be explained by assuming that the rate of the second electron supply to the oxygenated P-4501-substrate complex from cytochrome b5 is higher than that directly from NADPH-cytochrome P-450 reductase. An electron flow from NADH via cytochrome b5 can be utilized as the second electron for the O-deethylase reaction of 7-ethoxycoumarin catalyzed by reconstituted systems containing P-4502 and P-4482 when both NADH-cytochrome b5 reductase and cytochrome b5 are included in the system, although the cytochrome has no stimulatory effect at all on the deethylase activity of these two cytochrome P-450's. It has been shown that the second electron for P-4481 can also be supplied from NADH via cytochrome b5 in a reconstituted acetanilide p-hydroxylase system containing P-4481, cytochrome b5, and the two reductases.View full abstractDownload PDF (896K) -
Toshio TAKAGI1981 Volume 89 Issue 2 Pages 363-368
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMolecular weight of Aspergillus oryzae α-amylase (Taka-amylase A) was estimated to be 51, 000±500 by the combined use of high pressure silica gel (TSK-GEL G 3000 SW) chromatography and the low angle laser light scattering technique. The study was carried out partly to assess the performance of the combined technique, and results obtained indicate that it is highly promising as a method to determined protein molecular weight both accurately and quickly.View full abstractDownload PDF (1751K) -
Akihiro KIBE, Mariko FUKURA, Kenji KIHIRA, Taiju KURAMOTO, Takahiko HO ...1981 Volume 89 Issue 2 Pages 369-377
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS24-Nor-5β-cholestane-3α, 7α, 12α, 25-tetrol and 3α, 7α, 12α-trihydroxy-26, 27-dinor-5β-cholestan-24-one were administered intraperitoneally to bile fistula rats, and the metabolites excreted in the bile were analyzed. No formation of bile acids from these bile alcohols was observed. 7α, 12α, 25-Trihydroxy-24-nor-5β-cholestane-3α-O-(β-D-glucopyranosid) uronic acid was identified as the only biliary metabolite of the 24-nor-5β-cholestanetetrol. The major metabolite of the trihydroxy-26, 27-dinor-5β-cholestanone was 7α, 12α-dihydroxy-24-oxo-26, 27-dinor-5β-cholestane-3α-O-(β-D-glucopyranosid) uronic acid, and the minor metabolite was the glucurono conjugate of 26, 27-dinor-5β-cholestane-3α, 7α, 12α, 24β-tetrol. The results indicated that in rat liver these C25- and C26-bile alcohols, in contrast to C27-bile alcohols, were not converted into bile acids, and that the glucuronide production became necessary for hepatic elimination of the accumulated bile alcohols.View full abstractDownload PDF (735K) -
Toshihiko KONDO, Osamu TARUTANI, Nobuo UI1981 Volume 89 Issue 2 Pages 379-384
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe electrophoretic behavior of thyroglobulin and larger thyroglobulin-like iodo-proteins prepared from the hog thyroid was studied in polyacrylamide gels over a wide range of cross-linking degrees (C%). When examined at a constant total acrylamide monomer concentration (T%) of 5%., the mobility of 19S thyroglobulin decreased with increase in C below C=5%, whereas above C=5%, it increased markedly with increase in C, giving a minimum mobility at C=5%. Similar biphasic mobility curves were obtained with 27S and 37S thyroid iodoproteins. Furthermore, in a region above C=15%, at least two additional larger components which escaped detection in the gels with lower degrees of cross-linking appeared as separate bands. Ferguson plots constructed for thyroid iodoproteins at a higher constant C of 20%. gave straight lines intersecting at a common point at T=0%.. From the calculated slopes of the Ferguson plots, it has been established that the thyroid contains a series of multimers of 19S thyroglobulin as constituent iodoproteins. Structural parameters of highly cross-linked gels were estimated under the assumption that the gels would be predominantly composed of a random meshwork of gel beads.View full abstractDownload PDF (935K) -
Kazuyuki MORIHARA, Tatsushi OKA1981 Volume 89 Issue 2 Pages 385-395
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe peptide bond syntheses catalyzed by subtilisin BPN´, papain [EC 3. 4. 22. 2], and pepsin [EC 3. 4. 23. 1] were studied comparatively at the optimum pH of the enzymes with the coupling system Cbz-(AA)n-OH+Leu-X→Cbz-(AA)n Leu-X, in which AA=various amino acid residues, n=1-3 and X=-NH2, -OEt, -OBut, -ODPM (diphenyl methyl ester) or -NH_??_. The coupling with these enzymes differed depending upon the nature of (AA)n and X. For subtilisin-catalyzed coupling, the molecular size of the carboxyl component was most important. Thus, Cbz-Gly-Pro-Leu-OH was useful for synthesis in the presence of an equimolar concentration of Leu-NH_??_. Either Ala-NH2 or Leu-NH2 was also useful as an amine component when present at a concentration several times that of the carboxyl component. Papain catalyzed the coupling between Cbz-AA-OH (AA=glycine, L-alanine, L-valine, L-glutamic acid, or L-phenylalanine) and Leu-X (X=-OBut and -ODPM). The coupling of Cbz-Gly-Phe-OH and Leu-X catalyzed by pepsin was markedly affected, depending upon the nature of X in the following order: -NH_??_>-OBut>-NH2, -OEt. Leu-NH2, however, was quite efficient when its molar concentration was raised to twenty times that of the carboxyl component.View full abstractDownload PDF (798K) -
Kazumori MASAMOTO, Katsumi MATSUURA, Shigeru ITOH, Mitsuo NISHIMURA1981 Volume 89 Issue 2 Pages 397-405
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPartition of merocyanine dyes, which have a negative charge, onto photosynthetic membranes of chloroplasts and bacteria was analyzed by measuring the fluorescence intensity change, absorbance change, and amount of dye in the supernatant after centrifugation. The partition depended on the surface potential, which is a function of valence and concentration of ions in the medium.
The distribution of dyes between the membrane and aqueous phase was determined after centrifugation. The logarithm of the ratio of distribution was linearly related to the logarithm of salt concentration as predicted from the Gouy-Chapman theory and the Boltzmann distribution. Plots of the logarithm of fluorescence intensity against the logarithm of KCl and MgSO4 concentrations gave two straight lines with a slope ratio of about two. The absorbance change upon salt addition was also explained by the Gouy-Chapman theory.
The use of these dyes as probes of the surface potential of membranes is discussed.View full abstractDownload PDF (640K) -
Toshimi MIZUNUMA, Yukari TAKAHASHI, Hiromichi OKUDA1981 Volume 89 Issue 2 Pages 407-410
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of amino acids on insulin responsiveness in epididymal adipose tissue was examined. It was found that insulin-stimulated glucose oxidation in fat cells was significantly inhibited by glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, lysine, phenylalanine, and proline. The effect of insulin on glucose incorporation into triglyceride is also severely diminished by these amino acids. In addition, alanine reduced the incorporation of precursors ([U-14C]glucose or [1-14C]palmitate) into triglyceride both in vitro and in vivo. The K1 values of alanine were 0.4 and 0.5mM toward the precursors of glucose and palmitate, respectively. The mechanism of reduction of insulin responsiveness in rat adipose tissue is discussed on the basis of these results.View full abstractDownload PDF (270K) -
Shinsei MINOSHIMA, Masataka OHBA, Hiroshi HAYASHI1981 Volume 89 Issue 2 Pages 411-420
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn in vitro system for the methylation of methyl-accepting chemotaxis proteins (MCP's), which have been shown to be membrane integral proteins, was constructed. The system, consisting of the membrane, the cytoplasm, and labeled S-adenosyl methionine, showed the following characteristics.
1. The methylation of MCP in the membrane required the cytoplasm. The rate of incorporation of the labeled methyl group into MCP was dependent on the amount of the cytoplasm.
2. Incorporation of the labeled methyl moiety into MCP reached a steady state, and the level of the steady state incorporation was dependent on the concentration of the cytoplasm when the concentration of the membrane protein was constant.
3. The methyl moiety which had been incorporated into MCP before the steady state could be exchanged. It was suggested that the amount of methyl group introduced into MCP was equal to that of taken from MCP.
4. The methylated MCP was demethylated faster in the presence of a methyl donor than in its absence.
5. The membranes obtained from cheX-, cheB-, and cheZ mutants were inactive in the present in vitro system even when they were mixed with the wild type cytoplasm.View full abstractDownload PDF (735K) -
III. Amino Acid Sequence of a λ Type Bence Jones EuglobulinTatunori TAKAYASU, Nobuhiro TAKAHASHI, Tomotaka SHINODA, Tsuneo OKUYAM ...1981 Volume 89 Issue 2 Pages 421-436
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid sequence analysis has been done on a euglobulin-like λ Bence Jones protein NIG-58 with the major objective of determining the sequence of the variable region. Twenty-seven tryptic peptides with 4 overlapping peptides covering 215 residues, were isolated from completely reduced and aminoethylated protein, and 19 of these were completely sequenced. These comprised the entire variable region and 8 from the constant region. For the remaining peptides covering the rest of constant region, only partial sequences or the amino acid composition were determined. All the tryptic peptides could be arranged in order on the basis of the above results and homology with other λ chains of known sequences. The sequence of the variable region (residues 0-108) differed from those previously reported in 30 to 50 residues and was classified into the VλII subgroup, but no variation was found in the sequence of the last 105 residues. The protein is characteristic of euglobulin and has 2 additional half cystine residues at positions 26 and 28, which forms covalent polymers up to octamer when kept in an alkaline solution.View full abstractDownload PDF (2184K) -
Taiji IMOTO, Kiyotaka OKAZAKI1981 Volume 89 Issue 2 Pages 437-441
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and highly reproducible liquid chromatographic procedure was developed to fractionate peptides by using a prepacked reversed-phase column (RP-8) at low pressure. Peptides were eluted in 0.1% conc. HCl by using a gradient of ethanol in order of increasing hydrophobicity.View full abstractDownload PDF (293K) -
Hiroko YOSHIZAWA, Reiko UCHIMARU, Yoshio UENO1981 Volume 89 Issue 2 Pages 443-452
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nuclear biotransformation of aflatoxin B1 in vitro was observed with regard to inducer specificity, pH dependency, time course, kinetics, inhibitor sensitivity, and nuclear localization, and these data were compared with those from the microsomal transformation of aflatoxin B1. The nuclei and microsomes are capable of metabolizing aflatoxin B1 into aflatoxin M1, aflatoxin Q1, and two unidentified fluorescent compounds in the presence of fortified NADPH generating system. Pretreatments of rats by 3-methylcholanthrene or polychorinated biphenyl enhanced both the nuclear and microsomal C-9α-hydroxylation of aflatoxin B1 into aflatoxin M1 and phenobarbital or polychlorinated biphenyl induced aflatoxin Q1 production. The optimal pHs for aflatoxin M1 and Q1 were 8.3 and 7.4, respectively, both in the nuclei and microsomes. Kinetic analysis revealed the Km of aflatoxin M1 formation in methylcholanthrene-induced nuclei was 9.4×10-5M, and this value was very close to that obtained with the microsomes. Inhibitor experiments revealed a high sensitivity of aflatoxin M1 formation to 7, 8-benzoflavone and a low sensitivity of aflatoxin Q1 to SKF 525 A. These findings and data on the detergent treatment of nuclei suggest that the nuclear cytochrome P-448 system, induced by 3-methyl-cholanthrene and localized in the outer membrane, catalyzes the aflatoxin M1 formation, and the cytochrome P-450 system induced by phenobarbital biotransforms aflatoxin B1 into aflatoxin Q1.
Pretreatment of rats by phenobarbital was found to induce microsomal degradation or detoxication of aflatoxin B1 into water-soluble metabolites, and no such an induction was observed in the nuclei.View full abstractDownload PDF (1278K) -
Tetsu KAKUTANI, Hiroshi WATANABE, Kei ARIMA, Teruhiko BEPPU1981 Volume 89 Issue 2 Pages 453-461
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA copper-containing nitrite reductase was purified and crystallized from a potent denitrifying bacterium, Alcaligenes faecalis strain S-6. The enzyme was composed of 4 subunits with a molecular weight of about 30, 000, each containing 1 atom of Cu2+. Nitric oxide was identified as a main reduction product from nitrite in the enzyme-catalyzed reaction. The enzyme activity was inhibited strongly by KCN but only slightly by sulfhydryl reagents such as p-chloromercuribenzoate and N-ethylmaleimide.View full abstractDownload PDF (1626K) -
Tetsu KAKUTANI, Hiroshi WATANABE, Kei ARIMA, Teruhiko BEPPU1981 Volume 89 Issue 2 Pages 463-472
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA blue protein with a molecular weight of 12, 000 containing 1 atom of type I Cu2+ was purified and crystallized from a denitrifying bacterium, Alcaligenes faecalis strain S-6, as an inactivating factor for copper-containing nitrite reductase of the same organism. Inactivation of the enzyme occurred when the enzyme was incubated aerobically with a catalytic amount of the blue protein in the presence of reducing agents such as cysteine and ascorbate. The blue protein acts as a direct electron donor for the enzyme to catalyze the reduction of nitrite, but in the absence of nitrite, the enzyme-reduced blue protein system reacts with oxygen to produce H2O2. A suicide inactivation mechanism of the enzyme due to this H2O2 production is proposed.View full abstractDownload PDF (1639K) -
Takashi MURAMATSU, Hisako MURAMATSU, Masayuki OZAWA1981 Volume 89 Issue 2 Pages 473-481
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDolichos biflorus agglutinin (DBA), a lectin specific for N-acetylgalactosamine residues, agglutinated F 9 embryonal carcinoma cells. FITC-labeled DBA intensively stained F9 cells, and embryonal carcinoma cells and endodermal cells of teratocarcinoma OTT 6050, but did not stain a variety of cells from the host (129 mouse). Receptors for DBA were isolated from galactose-labeled F 9 and OTT 6050 cells by solubilization with 0.5% Triton X-100 and affinity chromatography on DBA-agarose. About 40% of the non-dialyzable galactose-label in the extracts of both types of cells was recovered in the receptors. On SDS gel electrophoresis, most of the receptors behaved as glycoproteins with molecular weights of more than 70, 000. The receptors from OTT 6050 cells contained about 0.8mg of carbohydrate per mg protein. Pronase digestion depolymerized the receptors from F 9 and OTT 6050 cells, but most of the resulting glycopeptides were still large enough to be excluded from a column of Sephadex G-50.View full abstractDownload PDF (1974K) -
Shigeru TAGUCHI, Hiroshi KOUYAMA, Nagasumi YAGO1981 Volume 89 Issue 2 Pages 483-489
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Acid β-galactosidase [EC 3. 2. 1. 23] of porcine adrenocortical lysosomes, assayed for its activity towards p-nitrophenyl-β-D-galactopyranoside, showed two activity peaks on gel filtration profile at pH 7.4, one corresponding to a molecular weight of approximately 270, 000 (termed form A 3) and the other about 65, 000 (termed form A 1).
2. Another form of acid β-galactosidase with a molecular weight of about 130, 000 (termed form A 2) was found when the high speed extract or partially purified form A 1 was chromatographed on Sephadex G-150 at pH 4.5.
3. In the presence of 0.1M NaCl or saturating amounts of substrate at pH 4.5, the high speed extract showed the aggregation of form A 2 yielding form A 3. Dissociation of form A 3 back to form A 1 was observed on incubation at 37°C in 0.02M sodium phosphate buffer, pH 7.4, and that was followed by irreversible enzyme inactivation.
4. Dissociation of form A 3 into form A 1 and enzyme inactivation in phosphate buffer, pH 7.4, were prevented by addition of 0.1M NaCl.
5. The interconvertible enzymic forms showed the same pH-activity profiles and Michaelis constants.
6. These results suggest that the lysosomal acid β-galactosidase in the porcine adrenal cortex exists in vivo as the dimer, and that the dimer may further aggregate into the tetramer.View full abstractDownload PDF (555K) -
Shiroki YAGISAWA1981 Volume 89 Issue 2 Pages 491-501
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzymatic method for converting tryptic peptides into their intermediates for the azide method of peptide synthesis was investigated by using model substrates, Bz-Arg, Bz-Gly-Arg, and Bz-Gly-Lys. The conditions favorite for the formation of the esters, hydrazides, and substituted hydrazides were deduced from the theoretical analysis on the pH and pKa dependence of the synthetic reactions and the pKa values of the substrates in various solvents. The theory based on the empirical relationship between the pKa of amines and the formation constant of the amides (Fersht, A. R. & Requena, Y. (1971) J. Am. Chem. Soc. 93, 3499-3504) indicates that the most favorite amine in the formation of amides is the one with a pKa value equal to the pKa of the carboxyl group and with such amines the amide formation takes place almost quantitatively even in aqueous solutions. Boc- and Cbz-hydrazine were found to satisfy the above condition approximately.
The methyl esters of the substrates were formed in 30-35% yields in 50% aqueous methanol at pH near 4.5 by the reverse reaction of trypsin [EC 3. 4. 4. 4]. Bz-Gly-Arg-NHNH2 was formed in a 34% yield in 2M aqueous hydrazine and in 70-80% yields in 50% aqueous solutions of dioxane, DMF, and DMSO containing 1M hydrazine at pH near 7. Bz-Gly-Arg-NHNH-Boc and Bz-Gly-Lys-NHNH-Boc were formed quite readily in 90-98% yields in H2O and in 50%. aqueous solutions of dioxane, DMF, and DMSO containing 0.5 or 1M Boc hydrazine at pH from 4 to 5. The Cbz-hydrazides of the peptides were also formed readily in 88-95 yields in the above mixed solvents containing 0.5M Cbz-hydrazine. The formation constants of the hydrazides in H2O were in good agreement with those predicted by the empirical relationship. The formation constants in the form of log Kθ in the mixed solvents calculated by taking the activity coefficients of the reactants as unity were 0.1 to 0.5 lower than the predicted values.View full abstractDownload PDF (846K) -
Takao TAKI, Yoshio HIRABAYASHI, Kuniaki TAKAGI, Ryoei KAMADA, Kiyohide ...1981 Volume 89 Issue 2 Pages 503-510
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple method for the preparation of oligosaccharide-linked aminohexyl-Sepharose 4 B (AH-Sepharose 4 B) and its application to the purification of anti-glycosphingolipid antibody which is specific for the oligosaccharide moiety are described. The oligosaccharide, which was obtained from galactosyl (β1→3) N-acetylgalacto-saminyl (β1→4) galactosyl (β1→4) glucosylceramide (asialo-GM1) by ozonolysis and subsequent alkali treatment, was covalently linked to the AH-Sepharose 4 B by reductamination in the presence of NaBCNH3. Anti-asialo-GM1 antibody was purified by means of an affinity technique with the oligosaccbaride-linked AH-Sepharose 4 B. The antibody bound to the affinity adsorbent was eluted with 0.5M NaSCN and 3.0M NaSCN. Antibody with higher specific activity was recovered in the 3.0M NaSCN fraction with 50% recovery of the activity of the starting material. The purified antibody was found to be quite specific for asialo-GM1. The presence of asialo-GM1 on the cell surface of free-type rat ascites hepatomas was confirmed by the immunofluorescence technique. The cell aggregates induced by the purified antibody were observed under a scanning electron microscope. The cell connection was found to occur at the tips of microvilli of the surface membrane. The localization of asialo-GM1 on the tips of the surface membrane was confirmed by means of the ferritin-conjugated antibody technique.View full abstractDownload PDF (2257K) -
Kenji IKEHARA, Harumi ANDO, Yoshimi TAKADA, Kin-ichi SUGAE1981 Volume 89 Issue 2 Pages 511-516
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExtracts of Bacillus subtilis vegetative cells with 2M formic acid contained a large amount of a hyperphosphorylated nucleotide (“spot 4” nucleotide). The compound always comigrated with authentic guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on two-dimensional polyethyleneimine (PEI)-cellulose thin-layer chromatography performed with three different solvent systems. Furthermore, all dephosphorylated 32P-labeled derivatives from the “spot 4” nucleotide comigrated on a one-dimensional PEI-cellulose plate with those from authentic ppGpp present in the same reaction mixture, when the compounds were hydrolyzed with snake venom phosphodiesterase or alkali. The level of the “spot 4” nucleotide (ppGpp) in the cell extracts was 0.14 nmol P/A660, corresponding to about one-third of the guanosine 5'-triphosphate (GTP) level and about 10%. of the adenosine 5'-triphosphate (ATP) level. These results indicate that a “magic spot” nucleotide, ppGpp, is present at a high level in B. subtilis cells vegetatively growing in mNSMP.View full abstractDownload PDF (1756K) -
Kenji IKEHARA, Kaeko MAEDA, Setsuko MAKINO, Kin-ichi SUGAE1981 Volume 89 Issue 2 Pages 517-521
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus subtilis was grown in a nutrient medium, mNSMP, and a synthetic medium, mS 6 (C), in which spore formation was initiated after vegetative growth and exhaustion of carbon source or glucose. The amounts of intracellular phosphorylated compounds were analyzed at intervals by 2M formic acid extraction and polyethyleneimine (PEI)-cellulose thin-layer chromatography followed by autoradiography. A hyperphosphorylated nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), was accumulated in cells during vegetative growth in both mNSMP and mS 6 (C), and then the nucleotide was degraded upon initiation of sporulation in both cases. Furthermore, after the nucleotide had disappeared in cells cultivated in mS 6 (C) upon exhaustion of the carbon source, it could be reformed in the sporulating cells by addition of glucose to the medium. These results suggest that the ppGpp in vegetative cells may function in the regulation of B. subtilis sporulation.View full abstractDownload PDF (1650K) -
Kazuhiko OTOGURO, Juichi AWAYA, Haruo TANAKA, Satoshi OMURA1981 Volume 89 Issue 2 Pages 523-529
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCell growth of Saccharomyces cerevisiae ATCC 12341 inhibited by the antibiotic cerulenin, a specific inhibitor of fatty acid synthesis, was restored by oleic acid (18:1) to give saturated fatty acid-starved cells, which could not grow when again transferred into a fresh synthetic medium containing the antibiotic and oleic acid. The growth of the saturated fatty acid-starved cells was restored when they were transferred into a medium supplemented with myristic acid (14:0), pentadecanoic acid (15:0), and palmitic acid (16:0) in the presence of cerulenin and oleic acid. Cellular saturated fatty acid content in the growth-restored cells was also restored to about two-thirds of that of the normal yeast cells. The DNA, RNA, and cell wall synthetic capabilities of the saturated fatty acid-starved cells were almost normal, but the L-leucine uptake and cytochrome pattern were severely impaired. These impairments were reversed on supplying palmitic acid. The decrease of L-leucine uptake of the yeasts was also caused by the addition of cerulenin alone. However, since the decrease occurred later than the inhibition of fatty acid synthesis, it was considered to be a secondary effect.
These results, obtained by using the saturated fatty acid-starved cells, indicate that the membranes of S. cerevisiae require certain amounts of saturated fatty acid and that the membrane functions (energy metabolism, transport, and so on) are impaired by starvation of saturated fatty acids.View full abstractDownload PDF (517K) -
Tetsuo OHMACHI, Kensuke SHIMURA1981 Volume 89 Issue 2 Pages 531-541
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Peptidyl-tRNA was prepared from the posterior silk gland ribosomes of Bombyx mori on the fourth to fifth days of the fifth instar to explore the initiation process in fibroin biosynthesis.
2. The peptidyl-tRNA was hydrolyzed at an alkaline pH and the resulting nascent peptides were fractionated on Sephadex G-75 and Sephadex G-200 columns into twelve fractions. Each fraction was analyzed for amino acid composition.
3. The nascent peptides of smaller molecular size were rather rich in glutamic and aspartic acids. However, the amino acid composition of the nascent peptides gradually approached that of fibroin as their molecular size increased.
4. A comparison between the nascent peptides of smaller molecular size and the small subunit of fibroin was made in respect to amino acid composition and tryptic peptide map. Considerable similarity between these two proteins was observed. The implications of these results in relation to the initiation process in fibroin biosynthesis are discussed.View full abstractDownload PDF (1241K) -
Fumie YAMASAKI, Hidenori HAYASHI1981 Volume 89 Issue 2 Pages 543-550
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe intracellular distribution of phosphodiesterase [EC 3. 1. 4. 17] induced by cyclic adenosine 3', 5'-monophosphate (cAMP) in Dictyostelium discoideum was studied. When cAMP-treated cells were homogenized and fractionated according to the method of de Duve et al. ((1955) Biochem. J. 60, 604), the specific activity of phosphodiesterase was highest in the light mitochondrial fraction. Peaks of specific activities of alkaline phosphatase (marker enzyme of membrane) and catalase (marker enzyme of peroxisomes) also appeared in the same fraction as phosphodiesterase. However, after centrifugation of the light mitochondrial fraction in a sucrose density gradient, the activity of phosphodiesterase was clearly separated with that of catalase (density 1.19g/ml) and showed three peaks at lower density (1.10, 1.13, 1.17g/ml) with good reproducibility. Some parts (1.13, 1.17g/ml) of the activity in the gradient overlapped with alkaline phosphatase activity, but in the density fraction of 1.10g/ml the activity of alkaline phosphatase was hardly detectable. When the light mitochondrial fraction was treated with Emulgen 108, or sonicated, phosphodiesterase was more easily solubilized than alkaline phosphatase and catalase, and was found in supernate after centrifugation at 20, 000×g for 30min. In order to distinguish the locations of the three enzymes, the supernatant of the light mitochondrial fraction treated with Emulgen 108 was subjected to charge shift electrophoresis. The electrophoretic mobilities of phosphodiesterase and catalase were unaffected by ionic detergent. However, alkaline phosphatase shifted towards the anode in the presence of anionic detergent (sodium deoxycholate), and shifted towards the cathode in cationic detergent (cetyltrimethylammonium bromide), relative to nonionic detergent (Emulgen 108) alone.
Thus, some part of the phosphodiesterase induced by cAMP may be associated with the plasma membrane, but the remainder is localized in some kind of intracellular particle of lower density. Moreover, the association with the membrane or particle is more easily dissociated than that of alkaline phosphatase, and the liberated phosphodiesterase is rather hydrophilic.View full abstractDownload PDF (583K) -
Tomoko KOZU, Tatsuya KURIHARA, Takeshi SENO1981 Volume 89 Issue 2 Pages 551-561
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree forms of DNA polymerase, named enzymes A, B, and C, that preferred (rA)n•(dT)12-18 as a template-primer, were partially purified from an extract of rat spleen. Enzymes B and C, both sedimenting at 9 S, appeared to correspond to DNA polyrnerase γ. However, they differed in their behavior on phosphocellulose and DNA-cellulose column chromatographies, and in their optimum KCl and divalent cation requirements for activity.
Enzyme A showed a unique property. Like DNA polymerase β, it sedimented at 3.8 S, was resistant to reagents blocking sulfhydryl groups, and was inhibited by phosphate, but it differed from DNA polymerase β with respect to elution positions from DEAE-cellulose, phosphocellulose and DNA-cellulose columns, Km value (lower by one order of magnitude for dTTP), and template-primer preference. Enzyme A was found in the mitochondrial fraction, in which DNA polymerase β was not detectable.
Enzymes A and C were isolated from the nuclear fraction, but this fraction did not contain enzyme B. The cytosol contained only enzyme A. The mitochondrial fraction contained enzyme A and enzyme C-like polymerase. Enzyme B was obtained with enzymes A and C only by extraction of the whole cell homogenate. Enzyme B may be labile or may be an artificial form of DNA polymerase γ formed during the purification procedures.View full abstractDownload PDF (725K) -
Yoshio UENO, Fumio TASHIRO1981 Volume 89 Issue 2 Pages 563-571
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical and biological properties of the hepatic metabolite of zearalenone, an estrogenic and non-steroidal fungal toxin produced by Fusarium species, were investigated by employing TLC, GC/MS, high pressure liquid chromatography and fluorospectral analyses, as well as uterine weight bioassay in immature mice. All the chemical and physical data supported the view that the major metabolite, obtained by incubating zearalenone with S-9 and microsomes of rat liver in the presence of NADPH, is C-6'-α-hydroxylated zearalenone (α-zearalenol). The estrogenic activity of this metabolite was several times higher than that of the parent zearalenone, and the results of biological and toxicological evaluations of α-zearalenol are discussed.View full abstractDownload PDF (559K) -
Effects of Triton WR-1339 on Lipoprotein Lipolytic Activity and Lipid Content of Rat Liver LysosomesHidenori HAYASHI, Shinkichi NIINOBE, Yoshio MATSUMOTO, Tetsuya SUGA1981 Volume 89 Issue 2 Pages 573-579
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe characteristics of lipoprotein lipolytic activity in lysosomes after the administration of Triton WR-1339 were studied, and the observed decrease in the density of the particles is discussed. The light mitochondrial fraction prepared from rat liver according to the method of De Duve et al. (Biochem. J. (1955) 60, 604) was used as a crude lysosomal fraction, in which acid phosphatase and lipoprotein lipase activities were concentrated. The lipoprotein lipolytic activity of lysosomes had a pH optimum of 4.0. The activity was strongly inhibited by Triton WR-1339 in vitro at low concentrations, while the acid lipase activity was almost unaffected, though relatively high concentration of the detergent significantly inhibited the latter activity. When Triton WR-1339 administered to rats (150mg/100g body weight), the activities of both the lipoprotein lipase and acid lipase of lysosomes from the treated rats decreased to one-third of those of control rats. Low-density and high-density lysosomes were partially purified from the light mitochondrial fraction from Triton WR-1339-treated and silver colloid-treated rats, respectively, by sucrose density gradient centrifugation. The low-density lysosomes (d=1.00-1.13) from Triton WR-1339 administered rats had approximately 4 and 3 times higher contents of triglyceride and cholesterol, respectively, than the high-density lysosomes (d>1.30) from silver colloid-treated rats.
In view of these results and the fact that the density of Triton WR-1339 is quite high (at least d=1.20), the decrease in density of hepatic lysosomes upon Triton WR-1339 administration cannot be due simply to incorporation of the detergent, and may rather be a result of incorporation and accumulation of some lipid(s) (possibly as lipoprotein) into lysosomes together with Triton WR-1339.View full abstractDownload PDF (1079K) -
Kunihiko KONNO, Ken-ichi ARAI, Mikiharu YOSHIDA, Shizuo WATANABE1981 Volume 89 Issue 2 Pages 581-589
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalcium binding to the regulatory light chain was studied by an equilibrium dialysis method, and it was found that calcium binding to the regulatory light chain in an isolated form was qualitatively different from that in a bound form, i.e., in myosin. This finding can acount for our previous observation (1979) that the calcium or strontium concentration required for inducing difference spectra in the regulatory light chain (in an isolated form) was higher than that required for activating ATPase or for superprecipitation of actomyosin (in a bound form).
Most of the findings obtained by Asada et al. (1979) and Ashiba et al. (1980) for clam foot myosin were confirmed with squid mantle myosin and scallop adductor myosin. Therefore, the following properties are probably not confined to clam foot myosin but are common to myosins from molluscan muscles. (1) The Mg-ATPase activity of myosin alone was sensitive to calcium. (2) Removal of the regulatory light chain resulted in a reversible loss of superprecipitation ability. (3) As the ATP concentration increased, the ATPase activity of actomyosin changed in a biphasic manner, whereas that of myosin alone changed in a monophasic manner.
Two of our observations appear to favor the suggestion of Asada et al. and Ashiba et al. that the primary action of calcium is to activate myosin-ATPase rather than to induce actin-myosin bindings. (1) Desensitized myosin (free from the regulatory light chain) behave exactly like untreated myosin in the presence of calcium. For example, as the ATP concentration increased, the ATPase activity of myosin alone and that of actomyosin changed qualitatively and quantitatively in the same way as those of untreated myosin and acto-untreated myosin in the presence of calcium. (2) At KCl concentrations between 0.2M and 0.4M, actin-activation of myosin (in the presence of calcium) was absent whereas calcium sensitivity of myosin-ATPase was still detectable.View full abstractDownload PDF (597K) -
Shigenori KANAYA, Tsuneko UCHIDA1981 Volume 89 Issue 2 Pages 591-597
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe purification procedure of ribonuclease T1 was greatly improved by introducing affinity chromatography with a new adsorbent, guanosine 5'-phosphate-aminohexyl-Sepharose 4 B. The enzyme was purified by only four steps with a high yield (68%) from Taka-Diastase powder. The purified enzyme preparation gave a single peak of protein with a small shoulder on DEAE-cellulose column chromatography. The peak fraction, amounting to approximately 90% of total proteins, was homogeneous ribonuclease T1. Moreover the shoulder fraction was shown to contain another form of ribonuclease T1 electrophoretically distinguishable from the original one. Comparison of the properties of the fraction containing almost equal amounts of both components with those of original ribonuclease T1 shows that the other form of T1 is identical with the original one in respect to amino acid composition and base specificity. We propose to designate this new form and original one as ribonuclease T1 -B and T1 -A, respectively.View full abstractDownload PDF (491K) -
Masaru SUZUKI1981 Volume 89 Issue 2 Pages 599-607
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDuring the course of studies on creatinine metabolism by bacteria, a newly isolated bacterial strain, Corynebacterium sp. U-96, showed the highest potency in the production of sarcosine oxidase. The enzyme was purified from the cell-free extract to a homogeneous state by successive procedures involving polyethyleneimine treatment and chromatographies on DEAE-cellulose, QAE-Sephadex A-50, DEAE-Sephadex A-50, Ultrogel AcA34, and creatine-AH-Sepharose 4B.
The absorption spectrum of the enzyme had maxima at 276, 369, and 451 nm, being characteristic of a fiavoprotein. This enzyme contains both 1 mol of non-covalently bound FAD and 1 mol of covalently bound FAD per mol of enzyme.
Sedimentation experiments gave an s020, W value of 9.35 and the molecular weight was calculated to be 174, 000 by the meniscus depletion method. The enzyme was shown to be composed of one each of four non-identical subunits; subunit A 110, 000; B 44, 000; C 21, 000; D 10, 000. One moleculk of FAD is covalently bound to subunit B.
The enzyme acted preferentially on sarcosine rather than on N-methyl-L-alanine or N-ethyl-glycine, but did not act at all on other N-methyl-L-amino acids tested. The Km and kcat values of the enzyme were estimated to be 3.4mM and 5.8 s-1 for sarcosine at pH 8.3 and 37°C, 8.7mM 2.1 s-1 for N-methyl-L-alanine at pH 8.3 and 37°C, and 11.4 mat and 2.2 s-1 for N-ethyl-glycine at pH 7.0 and 37°C, respectively.
The Km and Vmax values for oxygen as a hydrogen acceptor were found to be 0.13mM and 12.81 μmol/min/mg of enzyme. Oxygen can be replaced by 2, 6-dichlorophenolindophenol, phenazine methosulfate, ferricyanide, but not by methylene blue or cytochrome c.
The enzyme activity was strongly inhibited by Zn2+ Cu2+ Cd2+ Hg2+ Ag+ iodoacetamide, p-chloromercuribenzoate, or N-ethylmaleimide. Acetate, propionate, formaldehyde, and acetaldehyde also had inhibitory effects on the enzyme activity. The stoichiometry data showed that oxygen consumption was accompanied by the formation of equimolar amounts of formaldehyde, glycine, and hydrogen peroxide from sarcosine.View full abstractDownload PDF (1768K) -
Makoto SASAKI, Kimiyo YOSHIKANE, Etsuko NOBATA, Kenji KATAGIRI, Toshih ...1981 Volume 89 Issue 2 Pages 609-614
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe levels of two kinds of elastase-like enzymes, which are able to hydrolyze an artificial elastase substrate, suc-(Ala)3-pNA, but unable to hydrolyze a naturally occurring substrate, elastin, were found to be elevated in the sera of patients suffering from hepatobiliary disorders and other diseases accompanied by tissue damage. One of the enzymes was characterized as being sensitive to a chelating reagent, EDTA, and partially inactivated enzyme activity was recovered by the addition of calcium ion. The apparent molecular weight estimated by Sepharose 4 B column chromatography showed a wide distribution from 200, 000 to approximately 10, 000, 000, but all components were converted to a molecular weight of about 200, 000 by treatment with 2%. Triton X-100. The activity of this enzyme was partially reduced by the addition of anti-β-lipoprotein antibody, showing that a part of the enzyme was affiliated with low and very low density lipoproteins in the serum. The level of the other enzyme was rarely increased in the sera of patients suffering from severe hepatic disorders. This enzyme was resistant to EDTA, and the apparent molecular weight was 150, 000-200, 000. It appeared not to be associated with lipid component. Both enzymes were assumed to be tissue-derived enzymes, because their activities were very low in the sera of healthy persons.View full abstractDownload PDF (1050K) -
Tsutomu NOHNO1981 Volume 89 Issue 2 Pages 615-623
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe characteristics of the androgen receptor in the cytoplasmic fraction of Shionogi carcinoma 115 were studied in vitro by means of charcoal adsorption assay, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The equilibrium dissociation constant for [3H]dihydrotestosterone (Kd=(2-5)×10-11M) was estimated from independently determined rates of association and dissociation, and was lower by one order of magnitude than the value obtained by saturation analysis (Kd=(2-8)×10-10M). Evaluation of the effect of temperature on receptor binding of androgen allowed the estimation of several thermodynamic parameters, including activation energies of association (4 kcal/mol) and dissociation (14 kcal/mol), the apparent free energy (-13 kcal/mol), enthalpy (-9 kcal/mol), and entropy (+14 cal/mol per K). The receptor was greatly stabilized when bound with androgen. The results indicate how the lability of the unbound receptor and slow rate of dihydrotestosterone-receptor interaction can influence the estimation of dissociation constants by usual saturation analysis. The sedimentation coefficient of androgen receptor in freshly prepared cytosol was 6S, and became 7S after storage for 2 months at -80°C. The 7S conversion of the receptor was reversed by treatment with heparin. In all cases, a single 5S peak was obtained in the presence of 0.5M KCl. On electrophoresis in heparin-containing polyacrylamide gel, protein-bound radioactive androgen migrated as a single peak (Rf=0.5 in 5% gel). Differences in reported values for the sedimentation coefficient of androgen receptor in cytosol of Shionogi carcinoma 115 appear to be derived largely from aggregation of the receptor protein during the assay procedure.View full abstractDownload PDF (677K) -
Toshiyuki NAKADA, Tetsuya FUKUI, Terumi SAITO, Kazuyo MIKI, Chikako OJ ...1981 Volume 89 Issue 2 Pages 625-635
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSD(-)-β-Hydroxybutyrate dehydrogenase was purified from Zoogloea ramigera I-16-M to electrophoretic homogeneity. The molecular weight of the enzyme as determined by Sephadex G-200 gel filtration was 112, 000, and the monomer molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 28, 000, indicating that the native enzyme is a tetramer with four identical subunits. The enzyme showed a pH optimum at 8.0 in the oxidation reaction, and a broad pH optimum (5.5-7.5) in the reduction reaction. The Km values for D(-)-β-hydroxybutyrate and NAD in the oxidation reaction were 3.2×10-4M and 5.7×10-5M, respectively. The Km value for acetoacetate in the reduction reaction was 1.5×10-4M and that for NADH was 1.5×10-5M. Acetyl CoA, D-lactate, and 2-hydroxybutyrate were effective inhibitors for the oxidation of D(-)-β-hydroxybutyrate. The enzyme was sensitive to the inhibitory actions of sulfhydryl reagents such as p-chloromercuribenzoic acid, 5, 5'-dithiobis(2-nitrobenzoic acid) and HgCl2.View full abstractDownload PDF (1200K) -
Toshiyuki FUJIOKA, Kazutaka TANIZAWA, Yuichi KANAOKA1981 Volume 89 Issue 2 Pages 637-643
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSp-Amidinophenyl esters derived from a variety of amino acids and peptides, including D-amino acids, were synthesized. The kinetic behavior of trypsin towards these esters, which are “inverse substrates, ” was analyzed. Deacylation rates of acyl trypsins carrying D-amino acid residues were determined for the first time by the use of these “inverse substrates.” The steric requirements of the catalytic site region of trypsin were successfully analyzed by studying the deacylation process as manifested in the hydrolyses of enatiomeric pairs of “inverse substrates.” The effects of chiral ligands on the deacylation process were also studied in connection with the chiral requirements of the active site.View full abstractDownload PDF (509K) -
III. Novel Glycolipids from Turbo cornutusToshiko MATSUBARA, Akira HAYASHI1981 Volume 89 Issue 2 Pages 645-650
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive kinds of sphingoglycolipids were isolated from Turbo cornutus. Four of them were a series of novel glycolipids consisting only of galactose. The structures of these glycolipids were studied by methylation analysis, periodate oxidation, enzymatic degradation, and proton magnetic resonance spectroscopy. Three glycolipids were characterized as galactosyl(β1→1)ceramide, galactosyl(β1→6)galactosyl(β1→1)ceramide, and galactosyl(β→6)galactosyl(β1→6)galactosyl(β1→1)ceramide. Data indicating that the 4 th glycolipid might be the tetragalactosyl derivative of this series were obtained.
The carbohydrate moiety of the 5 th glycolipid, in contrast, was composed of fucose, galactose, glucose, and N-acetylglycosamine in a molar ratio of 1:2:1:1.View full abstractDownload PDF (409K) -
Shio MAKINO, Hiroshi NAKASHIMA, Keiko SHIBAGAKI1981 Volume 89 Issue 2 Pages 651-658
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine Band 3 was cleaved into two fragments by extracellular chymotryptic attack. As in the case of intact Band 3, both fragments were resistant to complete denaturation by guanidine hydrochloride and did not show the single cooperative conformational transition which is typical of many globular proteins. These results suggest that each Band 3 fragment contains several domains differing in resistance to denaturation. A discriminant function analysis further suggests that a considerable part of the polypeptide chain of the fragments is intercalated into or interacts with the lipid bilayer. These combined data are not incompatible with the suggestion of Drickamer ((1977) J. Biol. Chem. 252, 6909-6917) that the Band 3 polypeptide is probably folded to pass several times through the membrane.View full abstractDownload PDF (1220K) -
Tsukasa SEYA, Shigeharu NAGASAWA1981 Volume 89 Issue 2 Pages 659-664
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman C3 is composed of two disulfide-linked polypeptide chains, termed α chain (with a molecular weight of 110, 000) and β chain (with a molecular weight of 75, 000). When 3C was heated at above 50°C and at neutral pH, a single peptide bond in the α chain was selectively cleaved to yield two α chain fragments with molecular weights of 75, 000 and 44, 000. The two α chain fragments and intact β chain are originally connected by disulfide linkages but are gradually dissociated upon prolonged heat treatment. The dissociation seems to be caused by thiol-disulfide interchange reactions, since the dissociation was prevented by the addition of monoiodoacetic acid and only 1 mol of thiol group was determined to be newly generated upon heat treatment of C3. The heat-induced C3 cleavage reached a plateau when almost 1 mol of thiol group appeared. In addition, the heat-induced C3 cleavage was prevented by pretreatments with C3 convertase and methylamine, which are known to cleave the thioester linkage in the side chain of C3. Thus, the thioester linkage, which is the latent reactive site in C3 and which, upon activation of C3, forms an ester linkage with cell surfaces, seems to make a specific peptide bond extremely heat-labile.View full abstractDownload PDF (2319K) -
Motowo NAKAJIMA, Eriko TAMURA, Tatsuro IRIMURA, Satoshi TOYOSHIMA, Hir ...1981 Volume 89 Issue 2 Pages 665-675
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen chicken erythrocytes labeled with a stearic acid derivative spin label were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks due to the labels in membrane lipids, indicating an increase of membrane lipid fluidity. Addition of Con A increased the fluidity rapidly. This change was reversible only up to 30min after adding Con A, and thereafter it gradually became irreversible. However, if the erythrocytes were treated with cytochalasin B and methyl α-D-man-noside, a complete return of fluidity to the normal level could be observed at any stage after the binding of Con A. The observation of freeze-fracture replicas of erythrocyte membranes by transmission electron microscopy also showed that the redistribution of intramembranous particles gradually became irreversible after exposure to Con A. These results suggest that the microfilament-like system, which modulates the distribution of cell surface receptors for Con A, participates in the modulation of membrane fluidity.
Phospholipid methylation of chicken erythrocyte membrane was stimulated immediately after the binding of Con A. A methyltransferase inhibitor, 5'-deoxy-5'-S-isobutyl adenosine, abolished the increase of membrane fluidity within the first 10min and also that occurring later than 60min after adding Con A, but it was without effect on the elevated fluidity found between 20 and 60min. Removal of extracellular Ca2+ had an inhibitory effect on the lasting increase of fluidity. These results suggest that the first increase of membrane fluidity by Con A may be caused by phospholipid methylation, while the second increase may depend on the rearrangement of Con A receptor glycoproteins through cross-linking with Con A. The irreversible part of the membrane fluidity increase probably depends on Ca2+ influx, phospholipid methylation, and peripheral membrane proteins which constitute the microfilament-like system at the membrane inner surface.View full abstractDownload PDF (1976K) -
Teruo TANAKA, Naoko KAWANO, Tairo OSHIMA1981 Volume 89 Issue 2 Pages 677-682
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene of an extreme thermophile, Thermus thermophilus HB8, which codes for a leucine biosynthetic enzyme, 3-isopropylmalate (3-IPM) dehydrogenase [EC 1. 1. 1. 85], was cloned in Escherichia coli using pBR322 as a vector. E. coli cells carrying this recombinant plasmid, pHB2, produced the thermophilic enzyme 7-fold more than did T. thermophilus HB8 cells. When the crude extract of the pHB2-carrying cells was treated at 70°C for 10min, approximately 75% of the protein in the extract was precipitated with full activity of the thermophilic 3-IPM dehydrogenase being left in the supernatant, indicating that 4-fold purification was achieved during this process. This shows that the thermophilic 3-IPM dehydrogenase was purified 28-fold by these two procedures, cloning and heat treatment, and demonstrates that the extract from the plasmid-harboring cells is a good starting material for purification of the enzyme. Following the heat treatment, 3-IPM dehydrogenase was further purified by ammonium sulfate precipitation and DEAF-cellulose column chromatography. The enzyme preparation thus obtained contained 3-IPM dehydrogenase as a major component with a few minor impurities as shown by polyacrylamide gel electrophoresis, whereas the enzyme preparation from T. thermophilus HB8 cells obtained by the same procedures showed multiple bands on a polyacrylamide gel electrophoresis.View full abstractDownload PDF (1146K) -
Takeshi YAMAZAKI, Yousuke SEYAMA, Hideaki OTSUKA, Hideko OGAWA, Tamio ...1981 Volume 89 Issue 2 Pages 683-691
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMore than 90% of total lipids in the Harderian gland of guinea pig was identified as 1-alkyl-2, 3-diacylglycerol. The alkyl and acyl moieties consisted of saturated aliphatic chains ranging from 14 to 21 carbon atoms and from 15 to 26 carbon atoms, respectively. About 60 mol% of them had methyl branches, which were located at the even-numbered carbon atoms. Mono- and di-methyl branched chains accounted for most of the branched chains. The aliphatic chains contained methyl branches especially in the alkyl chains at the 1-position and the acyl chains at the 2-position.View full abstractDownload PDF (1873K) -
Kosuke MORIKAWA, Mitsushiro YANAGIDA1981 Volume 89 Issue 2 Pages 693-696
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method was developed to visualize individual DNA molecules in solution under a fluorescent microscope connected to a highly sensitive video camera. DNA stained with a fluorescent dye, DAPI, revealed thin extended filaments, thicker filaments and rapidly transforming folded structures dependent upon the solution conditions. Structural transitions were observed and recorded as video images. Our observations indicated the possibility that DNA has the ability of supercoiling itself. This DAPI staining method will have wide possible application in the study of DNA and chromatin.View full abstractDownload PDF (1409K) -
Yoh OKAMOTO, Takamitsu SEKINE1981 Volume 89 Issue 2 Pages 697-700
Published: 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was shown that the heavy chain structure was altered by the phosphorylation of light chain in gizzard myosin. Phosphorylated myosin was compared with unphosphorylated myosin with respect to the chymotryptic fragmentation with or without ATP in the medium. The rise and fall of the fragments were well explained by the reconstitution model for unphosphorylated myosin (Okamoto, Y., et al. (1980) J. Biochem. 88, 361-371). A specific cleavable site in the myosin head was completely abolished by the phosphorylation of light chain. The effect of phosphorylation on the site could be observed in the absence of ATP but not in its presence. These results strongly suggest the possible integrity of the site, 5 K daltons apart from the masked N terminus, for the physiological activity of gizzard myosin.View full abstractDownload PDF (774K)
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