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Volume 83 (1978)
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- Issue 5 Pages 1219-
- Issue 4 Pages 909-
- Issue 3 Pages 639-
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Volume 83, Issue 5
Displaying 1-34 of 34 articles from this issue
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Katsuhiko AKASHI, Kiyoshi KURAHASHI1978 Volume 83 Issue 5 Pages 1219-1229
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGramicidin A is an antibiotic peptide produced by Bacillus brevis ATCC 8185, which also produces tyrocidines. An attempt was made to establish a cell-free enzyme system for gramicidin A synthesis. An enzyme fraction, Component I, was partially purified from crude extracts of the organism and proven to be involved in the synthesis of the formyl-Val-Gly- region of gramicidin A. The initiation of gramicidin A biosynthesis is a function of Component I, which activates valine and binds it as a thioester, and further formylates it in the presence of formyltetrahydrofolic acid. The formylvaline thus synthesized is transferred to the glycine moiety, which is also thioesterified to Component I. Elongation of the peptide chain takes place by a mechanism similar to those found for tyrocidines, gramicidin S, and bacitracinView full abstractDownload PDF (2590K) -
Fuminobu YOSHIMURA1978 Volume 83 Issue 5 Pages 1231-1238
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSome properties of membrane ATPase activity in Veillonella alcalescens were examined. Mg2+ is required for the activity of the enzyme, and Ca2+ also activates the enzyme to some degree.
Of the nucleotide triphosphates, GTP and ITP were hydrolyzed to a lesser extent than ATP. The apparent Km for ATP hydrolysis was 0.25 to 0.60mM. ADP inhibited the enzyme and the kinetic data of its inhibition showed that the presence of ADP resulted in positive cooperativity. The enzyme activity was strongly inhibited by DCCD, azide, fusidic acid and the antibody to purified soluble ATPase from the thermophilic bacterium PS3.
Oligomycin, dinitrophenol, and ouabain showed no significant effect.View full abstractDownload PDF (558K) -
The Mode of Binding of 4-Thiouridylic Acid and a Fragment of Folic Acid, p-Aminobenzoylglutamic AcidKazuo TORII, Yukihide URATA, Yoichi IITAKA, Fumio SAWADA, Yukio MITSUI1978 Volume 83 Issue 5 Pages 1239-1247
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA four-Å electron density map was calculated for the monoclinic crystal of ribonuclease-S (RNase-S) based on two heavy-atom derivatives. Close geometrical similarity was found between the two crystallographically independent RNase-S molecules (called molecules ZA and ZB) in this crystal and that (called molecule Y) in the trigonal crystal. Using the rotational and translational parameters relating these three molecules, it was established that the crystal-lographic two-fold symmetry between the two molecules ZA in the monoclinic crystal was exactly identical to that between the two molecules Y in the trigonal crystal, suggesting the tendency of RNase-S molecules to associate in this way although the interaction is weak. The 4-Å difference Fourier maps calculated for the monoclinic crystal established the following conclusions. (1) 4-Thiouridine-2'(3')-monophosphate binds to the B1 and R1 sites like other pyrimidine nucleoside-2'(3')-monophosphates as expected from previous spectrophotometric studies, but not to the B2 site even at the concentration of 20mM. An attempt to visualize the photoproduct generated by irradiation of near-ultraviolet light in this complex failed. (2) p-Aminobenzoylglutamic acid, a fragment of folic acid, seems to bind to RNase-S with its benzene ring close to the B2 site and the α-carboxylate group close to the p1 site. The model is compatible with most of the chemical results obtained by Sawada et al. ((1977) Biochim. Biophys. Acta 479, 188-197).View full abstractDownload PDF (2045K) -
Yoko TANAKA, Takachika AZUMA, Kozo HAMAGUCHI1978 Volume 83 Issue 5 Pages 1249-1263
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pK values and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds in Bence Jones proteins and the Fab (t) fragment of a myeloma protein (Jo) (IgGI, K) were determined by means of the reactions with chloroacetamide and DTNB, and of spectrophotometric titration. The two thiol groups of partially reduced type K Bence Jones protein dimers had the same pK values (pK=9.76 at 0.2 ionic strength and 25°C) and the same true second-order rate constants (-K) toward chloroacetamide (-K=18.8×10-2M-1•S-1). The two thiol groups of partially reduced type λ Bence Jones protein dimers had different pK values but the variation of the pK values among the specimens was small (pK1=8.5-8.6 and pK2=9.5-9.7 at 0.2 ionic strength and 25°C). The spectrophotometric titration of partially reduced Nag protein (type λ) also showed that the two thiol groups have different pK values. The pK values of two thiol groups of the partially reduced Fab (t) fragment were determined as 8.51 and 9.76 at 0.2 ionic strength and 25°C. The effect of ionic strength on the pK values of the thiol groups of partially reduced Nag protein and the pK values of the thiol groups in partially reduced Ta protein (type K) and in a hybrid molecule formed between partially reduced Ta protein and partially reduced and alkylated H chains indicated that the difference in pK values did not arise from electrostatic interaction between the two thiol groups, but that the pK values are intrinsically different. The true rate constants, -K1 and -K2, of the two thiol groups of type λ Bence Jones proteins varied with the specimen (-K1=1.9-5.7×10-2M-1•S-1 and -K2=18.5-25.0×10-2M-1•S-1). The -K1 and -K2 values for Jo-Fab (t) were 7.21×10-2 and 23.1×10-2M-1•S-1, respectively. On the basis of these pK values and reactivities, we discuss the reformation of the interchain disulfide bonds from partially reduced Bence Jones proteins and immunoglobulins in the presence of oxidized glutathione.View full abstractDownload PDF (1079K) -
I. Preparation and CharacterizationHidenori NARUSE, Hikoichi SAKAI1978 Volume 83 Issue 5 Pages 1265-1273
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new inhibitory factor of the microtubule (MT) assembly system was isolated from unfertilized sea urchin egg cortex. This factor not only suppressed spontaneous brain MT assembly, but also induced depolymerization of the reconstituted MTs. The factor did not suppress initial MT growth initiated by ciliary outer fiber fragments but the assembled MTs were soon depolymerized with time. The inhibitory activity was heat-stable but sensitive to trypsin or urea. The mode of the inhibition was distinct from the inhibitory effects of RNA on the MT assembly. The inhibitory factor partially purified on DEAE-Sephadex A-50 completely inhibited tubulin polymerization in a factor: tubulin ratio of 0.013.View full abstractDownload PDF (567K) -
Haruhiko TAKISAWA, Yuji TONOMURA1978 Volume 83 Issue 5 Pages 1275-1284
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of Ca2+, Mg2+-dependent ATPase [EC 3.6.1.3] of fragmented sarcoplasmic reticulum (FSR) was studied in the presteady state, and the following results were obtained.
1. The P1 burst reported by Kanazawa et al. ((1971) J. Biochem. 70, 95-123) was caused by the transition of v/[EP] after the EP overshoot and the P1 burst observed by Froehlich and Taylor ((1975) J. Biolchem. 250, 2013-2021) had occurred.
2. When the reaction was started by adding Ca2+ and [γ-32P]ATP to FSR or [γ-32P]ATP to FSR preloaded with Ca2+, neither the E32P overshoot nor the 32P1 burst were observed.
3. The time course of P1 liberation showed a lag phase and a burst phase, and the apparent rate constant of EP decomposition (v/[EP]) showed a very complicated pattern during the initial phase of the reaction. It increased from the initial value, reached a maximum, then decreased to the steady-state level. This phenomenon was observed more clearly at 4°C than at 20°C.
We also studied the kinetics of solubilized SR ATPase, and obtained the following results.
1. The SR ATPase in the solubilized state showed neither the EP overshoot nor the P1 burst.
2. The double reciprocal plots of the ATPase activity and the amount of EP in the steady state against the ATP concentration gave straight lines over a wide ATP concentration range.
3. The amount of ADP bound to the enzyme during the ATPase reaction was estimated by measuring the amount of ADP remaining in the SR ATPase-ATP system coupled with creatine kinase (CK) [EC 2.7.3.2] and creatine phosphate (CP). The amount of bound ADP was found to be negligibly small.
We thus concluded that the EP overshoot and the P1 burst which coincided with the transient decay of EP during the initial phase are not caused by the formation of an acidlabile intermediate (E•P) as Froehlich and Taylor suggested, but depend on the enzyme state in the membrane, probably on the interactions of the ATPase with phospholipids (PL). Furthermore, the acceleration of the ATPase reaction by ATP also depends on the enzyme state in the membrane, and the EP intermediate does not contain bound ADP at least in the steady state of the ATPase reaction.View full abstractDownload PDF (735K) -
Yukio YOKOTA1978 Volume 83 Issue 5 Pages 1285-1292
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis.
By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200, 000. The subunit size was found to be 50, 000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure.
The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.View full abstractDownload PDF (1107K) -
Yukio YOKOTA1978 Volume 83 Issue 5 Pages 1293-1298
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells.
Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290, 000. The molecular weight of the subunit was estimated to be 72, 000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72, 000.View full abstractDownload PDF (386K) -
Morio SETAKA, Tatuhiko ICHIKI, Hiroshi SHIMIZU1978 Volume 83 Issue 5 Pages 1299-1303
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rates of uptake and release of 2, 2, 6, 6 tetramethyl piperidinyl-l-oxycholine (Tempo-choline) for vesicles made of dipalmitoyl phosphatidylcholine (DPPC) and of egg phosphatidylcholine-cholesterol mixtures were measured by ESR and found to have interesting temperature-dependences. In the former case, both rates exhibit a sharp maximum at the critical temperature of phase transition of the bilayer membrane. In the latter case, the permeability of the membrane to Tempo-choline is asymmetric with respect to uptake and release: uptake is appreciable at temperatures higher than 66°C, while release is observable only at temperatures higher than 80°C. The asymmetric permeability is explained in terms of the asymmetric distribution of cholesterol between the outer and inner membranes of the vesicle.View full abstractDownload PDF (375K) -
Sadao WAKABAYASHI, Toshiharu HASE, Keishiro WADA, Hiroshi MATSUBARA, K ...1978 Volume 83 Issue 5 Pages 1305-1319
Published: 1978
Released on J-STAGE: April 16, 2015
JOURNAL FREE ACCESSThe amino acid sequences of two ferredoxins isolated from pokeweed , Phytolacca americana, were determined. Tryptic peptides of maleyl-carboxymethyl-ferredoxin I and carboxy-methyl-ferredoxin II were prepared and analyzed. The large peptides were further digested with staphylococcal protease and chymotrypsin. Ferredoxins I and II were composed of 96 and 98 amino acid residues, respectively. Though ferredoxin I lacks tryptophan and methionine, ferredoxin II contains both of them. In a comparison of the amino acid sequences with those of other higher plant ferredoxins, ferredoxin I is one residue shorter than others at the carboxyl-terminus and ferredoxin II one longer than others at the amino-terminus. Ferre doxins I and II differ in 23 sites from each other and in 27 to 37 sites from other higher plant ferredoxins. This suggests that duplication of the ferredoxin gene occurred after the divergence of pokeweed from other higher plants. A phylogenetic tree including all other ferredoxins was constructed.View full abstractDownload PDF (2190K) -
Toshiharu HASE, Sadao WAKABAYASHI, Hiroshi MATSUBARA, Michael C. W. EV ...1978 Volume 83 Issue 5 Pages 1321-1325
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have determined the amino acid sequence of a ferredoxin from a photosynthetic green sulfur bacterium, Chlorobium thiosulfatophilum strain Tassajara. It contains 61 amino acid residues with 9 cysteines, and 8 of the 9 were located at positions corresponding to those in clostridial-type ferredoxins. Other structural features were closer to those of ferredoxins from another photosynthetic bacterium, C. limicola, than to those of non-photosynthetic bacteria. Compared with ferredoxin from Chromatiwn, a photosynthetic purple sulfur bacterium, all photosynthetic bacterial ferredoxins have a common region in the carboxyl-terminal half with several extra residues and a unique cysteine residue. We compared all the photosynthetic bacterial ferredoxins that have been sequenced and concluded that C. thiosulfatophilum ferredoxin is most closely related to C. limicola ferredoxin I.View full abstractDownload PDF (320K) -
Tetsuya ASAKAWA, Michiko HASUNUMA, Fumi MORITA1978 Volume 83 Issue 5 Pages 1327-1335
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEnzymatic properties of heavy meromyosin (HMM) were studied using ribose 5'-triphosphate (RTP) and tripolyphosphate (TP) as substrates. The maximum value of the steady state rate of RTPase depended on the kind of divalent cation added. The pH-activity curve of RTPase and its transition with temperature were similar to those of ATPase. The UV-absorption difference spectrum of HMM due to tryptophanyl residue was induced by RTP and showed decaying as in ATPase. 1.2 mol of P, -burst/mol of HMM was observed with the RTPase reaction. All these results are similar to those of ATPase. The major intermediate at the steady state of RTPase at a higher temperature, therefore, may be one corresponding to EADPp(α) which shows the ATP-form of the difference spectrum in ATPase. However, the maximum rate of steady state RTPase in the presence of MgCl2 was about 10 times larger than that of ATPase. This result suggests that the adenine ring of the ATP molecule has a role in elongating the life time of the EADPp(α) state. On the other hand, the maximum rate of steady state TPase was scarcely affected by the added divalent cation and was about one fourth the maximum rate of Mg2+ ATPase. No P1-burst was detected in the TPase reaction. These results suggest that the rate determining step of TPase is the cleavage step. The cleavage rate seems to be far smaller than that of RTPase. The ribose moiety of the ATP molecule is supposed to play a role in enhancing the cleavage. F-Actin activated the HMM-RTPase only three times. Both superprecipitation of actomyosin and shortening of glycerinated psoas fiber were not induced by either RTP or TP. On the basis of these results, it was concluded that both the adenine and ribose moieties of the ATP molecule have roles in elongating the life time of the EADPp(α) complex. The conditions showing a longer life time of the complex in the absence of F-actin may be necessary to cause muscle contraction.View full abstractDownload PDF (640K) -
Hiroshi KANAZAWA, Yasuhiro ANRAKU1978 Volume 83 Issue 5 Pages 1337-1343
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of glucose and glucose-6-phosphate in initiating the repression of, β-galactosidase synthesis were studied using a mutant of Escherichia coli K12 which lacks glucose-specific enzyme II of the phosphoenolpyruvate-sugar phosphotransferase system. It was found that glucose-6-phosphate causes transient repression of β-galactosidase synthesis but glucose does not cause transient repression in this mutant.
Evidence was obtained that both the presence of an active transport system for glucose-6-phosphate in the cells and glucose-6-phosphate in the medium are necessary for the initiation of transient repression. No metabolism of glucose-6-phosphate is required. Upon depletion of glucose-6-phosphate in the medium the transient repression was reversed. After the reversal the rate of enzyme synthesis was high in the cells which had been exposed to a high concentration of glucose-6-phosphate. It was concluded that the translocation of glucose-6-phosphate across the membranes is the primary event which affects both the initiation of and the recovery from the transient repression. During the transient repression the cellular content of cyclic adenosine 3', 5'-monophosphate decreased significantly.View full abstractDownload PDF (496K) -
Daisuke TSURU, Kunio KADO, Kunio FUJIWARA, Mikio TOMIMATSU, Kiyokazu O ...1978 Volume 83 Issue 5 Pages 1345-1353
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interactions of porcine α2 macroglobulin (α2M) with native proteinases, their zymogens and the chemically-modified enzymes were compared. The α2M did not bind to chymo-trypsinogen, or to most of the chemically modified derivatives of α-chymotrypsin, trypsinogen, DIP- and PMS-trypsins, but it could interact with anhydrotrypsin, PMS-subtilisin, and 0-acetylated neutral subtilopeptidase. Anhydrotrypsin appeared to bind very tightly to α2M, as does native trypsin, whereas the binding of PMS-subtilisin to α2M was weaker than that of the native enzyme, judging from exchange experiments with labeled enzyme and from competitive enzyme assay.
There are, however, some differences in the mode of interaction with α2M between native and anhydrotrypsins. (1) The shape and the magnitude of ultraviolet difference spectra caused by the interaction with α2M were significantly different. (2) The interaction of α2M with active proteinase led to the formation of new amino-terminal amino acids, while that with anhydrotrypsin did not. (3) In vivo experiments showed that radioactivity of 3H-labeled trypsin-α2M complex was rapidly cleared from the plasma of rats, whereas the anhydrotrypsin-α2M complex was cleared very slowly. These results suggest that the proteolytic activity of the enzyme is not obligatory for the first phase of α2M-proteinase interaction (formation of a Michaelis-type complex), but only the proteolytically modified complex is cleared rapidly from the blood circulation system.View full abstractDownload PDF (696K) -
Nobumasa YASOGAWA, Yukihiro SANADA, Nobuhiko KATUNUMAI1978 Volume 83 Issue 5 Pages 1355-1360
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ability of serine protease of skeletal muscle to degrade native myofibrillar proteins, such as myosin, actin, troponin, tropomyosin, α-actinin, and M-protein from rabbit skeletal muscle was studied. The amino acids or peptides liberated from these proteins by the protease were determined fluorometrically using o-phthalaldehyde. The order of their susceptibilities at a molar ratio of the serine protease to substrate of 1:100 was: myosin>>troponin>tropomyosin>actin. α-Actinin and M-protein were not degraded. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis showed that the myosin heavy chain was degraded into two fragments, having molecular weights of 100, 000 and 88, 000, whereas the light chains were scarcely degraded. The serine protease degraded troponin-T rapidly and troponin-I slowly, but did not degrade troponin-C. Tropomyosin was degraded rapidly into two components with molecular weights of 21, 500 and 19, 000. Actin was degraded slowly, but no liberated fragment could be detected.View full abstractDownload PDF (1481K) -
Takashi OSUMI, Takashi HASHIMOTO1978 Volume 83 Issue 5 Pages 1361-1365
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of di-(2-ethylhexyl)phthalate (DEHP) administration on cyanide-insensitive palmitoyl-CoA oxidizing activity in liver was studied. Two weeks of DEHP treatment increased the activity by one order of magnitude in male Wistar rats. A similar effect was also observed in male and female Sprague-Dawley rats and mice, but not in guinea pigs.
When the liver was fractionated by differential centrifugation, the activity was concentrated in the light mitochondrial fraction. On the subfractionation of this fraction by sucrose density gradient centrifugation, the activity was distributed in a pattern similar to that of urate oxidase, but not resembling that of glutamate dehydrogenase. These data suggest that a fatty acyl-CoA oxidizing enzyme system which is located in peroxisomes is induced by the administration of DEHP.View full abstractDownload PDF (390K) -
Takenori YAMADA, Junji SAGARA, Hiroshi SHIMIZU1978 Volume 83 Issue 5 Pages 1367-1374
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA “ghost” myofibril (myosin-extracted myofibril) Sephadex conjugate which specifically binds myosin, HMM and S-1 in the absence of Mg-ATP or Mg-PP1 can be prepared in a few days by conjugating “ghost” myofibrils to Sephadex beads. Binding ability is retained for over a month. It is used, therefore, for actin-affinity chromatography of myosin and its active fragments.
It is under debate whether the two heads of the myosin molecule are functionally identical. Recently several reports have indicated that S-1 could be separated into two kinds of S-l, one giving the initial burst of phosphate and the other not, by assuming a difference in the affinity of the two kinds of S-1 to F-actin. Attempts are reported here to obtain these two components of S-1 separately by using the “ghost” myofibril Sephadex conjugate column. The method of S-1 separation reported by Shibata-Sekiya and Tonomura ((1976) J. Biochem. 80, 1371-1380), which used S-1 treated with CMB, was applied to the “ghost” myofibril Sephadex conjugate column. This resulted in the successful separation of S-1 modified with CMB giving no initial burst of phosphate and unmodified S-1 giving the initial burst of phosphate. A eparation method based essentially on the principle employed by Taniguchi and Tawada ((1976) J. Biochem. 80, 853-860) gave an unsuccessful result.View full abstractDownload PDF (1890K) -
Yoh OKAMOTO, Takamitsu SEKINE1978 Volume 83 Issue 5 Pages 1375-1379
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTryptic digestion of gizzard myosin resulted in the degradation of the 20K light chain (G1) to its 17K fragment, which could not be phosphorylated. The rapid loss of Ca2+-dependent activa-tion of actomyosin ATPase activity accompanied the degradation of G1. Increase in the Ca2+-ATPase activity and decrease in the EDTA-ATPase activity of myosin accompanied the degradation of myosin heavy chain, but not the cleavage of G1.View full abstractDownload PDF (1275K) -
Yoshifumi HORIUTI, Shigeyuki IMAMURA1978 Volume 83 Issue 5 Pages 1381-1385
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFatty acids prevented adsorption of purified Chromobacterium lipase [triacylglycerol acylhydrolase, EC 3.1.1.3] onto palmitoyl cellulose (Pal-C) and also increased the activity of the purified lipase. These effects increased with increase in the concentration and chainlength (up to 16 carbon atoms) of the fatty acids, and long-chain unsaturated fatty acids, such as oleic acid, linoleic acid and erucic acid, were most effective.
When the lipase was adsorbed (immobilized) on Pal-C, its activity was elevated to 20-times that of the free lipase in detergent-free reaction mixture (olive oil-buffer system). Thus lipase was adsorbed to Pal-C through a hydrophobic site distinct from its catalytic site and the binding of fatty acids to the hydrophobic site seems to result in stimulation of the lipase activity.View full abstractDownload PDF (353K) -
V. Comparative Studies of Malic Enzymes in BacteriaMasahiro IWAKURA, Masanobu TOKUSHIGE, Hirohiko KATSUKI, Shigeru MURAMA ...1978 Volume 83 Issue 5 Pages 1387-1394
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSScreening of four malic enzymes-NAD-linked enzyme [EC 1.1.1.38], NAD, NADP-linked enzyme [EC 1.1.1.39], NADP-linked enzyme [EC 1.1.1.40], and D-malic enzyme was carried out with cell-free extracts of the following 16 strains of bacteria by the aid of Sepharose 6B column chromatography: 9 strains of enteric bacteria, 3 strains of Pseudomonas, Alcaligenes faecalis, Agrobacterium tumefaciens, Rhodospirillum rubrum, and Clostridium tetanomorphwn. All the strains tested contained at least one malic enzyme. The NADP-linked enzyme activity was found in all the strains except C. tetanomorphaon, the NAD-linked enzyme activity in 12 strains-8 strains of enteric bacteria, 2 strains of Pseudoinonas, Ag. tumefaciens, and C. tetanomorphum-and D-malic enzyme activity in 4 strains-A, aerogenes (IFO 3319 and 12059), Ps. fluorescens, and R. rubrum. The NADP-linked and NAD-linked enzyme activities of two strains of Pseudomonas were not separated by the chromatography. The available evidence suggested that the NAD, NADP-linked enzyme was not present in these 16 strains.
The comparative studies of molecular, enzymatic, and serological properties of the malic enzymes in these 16 strains revealed a close similarity of the same types of malic enzymes among enteric bacteria.View full abstractDownload PDF (1760K) -
Hideo MISAKI, Makoto MATSUMOTO1978 Volume 83 Issue 5 Pages 1395-1405
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaenzolyticius with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis.
The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89, 000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15, 000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis.
The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all.
The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate.
The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37°C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.View full abstractDownload PDF (1339K) -
Motohiro TSUJI, Teruo NAKAJIMA1978 Volume 83 Issue 5 Pages 1407-1412
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation of γ-aminobutyric acid (GA-BA) from putrescine was examined in organs of rats using radioactive putrescine. Radioactive GABA was detected in all the organs of a rat injected intraperitoneally with radioactive putrescine, and the highest radioactivity of GABA was observed in the small intestine. The enzyme involved in this formation was purified from small intestine and identified as a diamine oxidase, histaminase, from the properties of the enzyme. Activity of the enzyme was found in all the organs of rat, and the highest activity was observed in the small intestine.View full abstractDownload PDF (454K) -
Hikoichi SAKAI, Gen MATSUMOTO1978 Volume 83 Issue 5 Pages 1413-1422
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSDS-polyacrylamide gel electrophoresis of axoplasmic proteins and viscometric measurements of axoplasmic tubulin polymerization were performed using giant axons of the squid, Doryteuthis bleekeri. The axoplasm contained major protein species having apparent chain weights of 200, 000, 78, 000, 59, 000, 52, 000, 49, 000, and 43, 000 daltons as well as high molecular weight components. The 200, 000 dalton component and some of the 59, 000 and 52, 000 dalton components were found in the pellet after high speed centrifugation. Axoplasmic microtubule (MT) proteins were obtained by one cycle of temperature-dependent polymerization and depolymerization. SDS-polyacrylamide gel electrophoresis showed that the squid MT protein fraction consists mostly of tubulin and high molecular weight proteins but lacks the component corresponding to the major high molecular weight protein usually detected in porcine brain MT proteins. The α and β subunits of squid tubulin were shown to have the same mobilities as those of porcine brain tubulin. The viscometric measurements indicated that the optimal temperature for MT assembly was 25°C and optimal salt concentrations were 0.1M for KCl and KF, and 0.3M for K glutamate. The ability of K halides to support assembly was in the order F->Cl->Br->I-. I- was totally inhibitory for assembly. The assembled axo-plasmic MT's were sensitive to SH-blocking reagent, Ca ions, colchicine, and cold, in the same way as porcine brain MT's. From axoplasm-extruded axons, a high molecular weight protein was extracted with 0.6M KI or 10mM CaCl2. This component was shown to be capable of promoting the polymerization of porcine brain tubulin dimers.View full abstractDownload PDF (3204K) -
Hitoshi UENO, Tatsuo OOI1978 Volume 83 Issue 5 Pages 1423-1433
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit skeletal α-tropomyosin, separated by hydroxyapatite chromatography, was treated with trypsin (1/100 wt/wt) at 0°C for 24h. Trypsin-resistant fragments of tropomyosin were separated into the precipitate and supernatant fractions at pH 4. 3 in 1M KCl, and these were subjected to QAE-Sephadex A50 column chromatography for further purification. SDS-gel electrophoresis showed 16, 000 and 14, 000 dalton bands for the supernatant (s-fragment) and an 11, 500 dalton band for the precipitate (p-fragment). We obtained a 13, 500 dalton chain (13, 500 dalton fragment) in addition to the s- and p-fragments upon treatment with more dilute trypsin (1/500 wt/wt) for 48h at 0°C. Both the p- and 13, 500 dalton fragment had the same C-terminal portion as intact α-tropomyosin, and could form an intra-chain disulfide bond on oxidation. Therefore, these two fragments were deduced to be polypeptides from some points on the N-terminal side of Cys 190 to the intact C-terminal. The s-fragment, on the other hand, did not contain any cysteine, Phe, or His residues according to amino acid analysis, suggesting that the fragment is derived from the N-terminal side from Cys 190. Tentative assignment of the fragments was carried out by amino acid analysis, and C- and N-terminal determination.
The p-, s-, and 13, 500 dalton fragments appear to be in coiled-coil form in solution, having α-helical contents of 77, 71, and 64%, respectively, and are able to interact with intact tropomyosin to reduce the viscosity of tropomyosin solution.
The s-, p-, and 13, 500 dalton fragments have little binding capacity individually to troponin, but the mixture, i.e., the s- and p-fragments, the 13, 500 dalton fragment and the N-chain, which was obtained by cleavage at Cys 190, showed clear binding with troponin independent of Ca2+ in solution as detected by gel electrophoresis. The p-fragment showed some binding to troponin, since cross-linkage to troponin was possible by treatment with dimethyl suberimidate.
From the result, it can be inferred that the troponin binding regions in tropomyosin are located on both sides of Cys 190, where trypsin attacks more easily than at other parts of the molecule, leaving two trypsin-resistant fragments.View full abstractDownload PDF (2453K) -
Toshiko KUNIKATA, Yasunori NITTA, Takehiko WATANABE1978 Volume 83 Issue 5 Pages 1435-1442
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The difference spectra of Taka-amylase A [EC 3. 2. 1. 1] of Aspergillus oryzae produced bythree substrates (α-cyclodextrin, maltotriose, and maltose) showed a clear peak at 293 nm due to the red shift of the tryptophan residues. The dependency of the molar extinction coefficient, Δε, of the difference spectrum at 293nm on the substrate concentration was investigated at 25°C and pH 5.3 (0.08M acetate buffer). From the titration of Δε with substrate, the dissociation constant, K, of the enzyme-substrate complex and the value of the maximum change in the molar extinction coefficient for saturation of the enzyme with substrate, ΔεBmax, were determined as follows: values of Kwere 3.5mM, 35mM, and 55mM, and those of ΔεBmax were 1, 970, 1, 410, and 730 for α-cyclodextrin, maltotriose, and maltose, respectively. For each substrate, the value of K was in good agreement with that of the Michaelis constant, Km, or inhibitor constant, K1.
2. The difference spectrum produced by ethylene glycol, a nonspecific substance with respect to this enzyme, had a maximum peak at 287nm, and lacked a clear peak at 293nm. When this enzyme was almost saturated with α-cyclodextrin (about 77% saturation), the difference spectrum with ethylene glycol decreased in the range of wavelength from 280nm to 310nm. The difference between the two difference spectra with ethylene glycol was obtained as a spectrum. The spectrum showed a clear peak at 293nm and the molar extinction coefficient of this peak was about 1, 000 per 20% ethylene glycol.
3. From the results, we estimated that about three tryptophan residues accessible to solvent exist in the substrate binding site of this enzyme, and that these residues become inaccessible to solvent when the enzyme-substrate (α-cyclodextrin) complex is formed.View full abstractDownload PDF (1114K) -
XIII. Preparation of 6-Deoxy-6-Iodomaltooligosaccharides and Their Inhibitory Action against Taka-Amylase A1Kaoru OMICHI, Keiko FUJII, Teruo MIZUKAMI, Yoshio MATSUSHIMA1978 Volume 83 Issue 5 Pages 1443-1447
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSO-α-D-Glucopyranosyl-(1→4)-O-6-deoxy-6-iodo α-D-glucopyranosyl-(1→4)-D-glucopyranose (6'-MT), O-α-D-glucopyranosyl-(1→4)-6-deoxy-6-iodo-D-glucopyranose (6-M), and O-6-deoxy-6-iodo-α-D-glucopyranosyl-(1→4)-D-glucopyranose (6'-M) were prepared and their inhibitory action against Taka-amylase A [EC 3.2.1.1, α-1, 4-glucan 4-glucanohydrolase, Aspergillus oryzae] was investigated. The inhibitor constants of 6'-MT and 6'-M were 10mM and 54mM, respectively, and both inhibitors showed mixed-type inhibition. 6-M scarcely inhibited the enzyme action.View full abstractDownload PDF (960K) -
Satoshi NASU, Kiyokazu SHIOJI, Iwao UEDA, Yoshinori TANIGAWA, Makoto S ...1978 Volume 83 Issue 5 Pages 1449-1458
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+/protein modulator-dependent and -independent guanosine 3':5'-monophosphate (cGMP) phosphodiesterases were separated from hog heart. The protein modulator-free Ca2+/protein modulator-dependent enzyme was partially purified by repeated DEAF-cellulose column chromatography and heat treatment. The final preparation of this enzyme showed no significant basal activity under the standard assay conditions.
Lineweaver-Burk plots of the Ca2+/protein modulator-dependent enzyme activity indicated the presence of only a single kinetic form of the enzyme with Km=2.0×10-6M for cGMP, whereas the plots for the independent enzyme were anomalous, showing both high and low Km values for cGMP.
The Ca2+/protein modulator-dependent enzyme proved relatively stable at 48°C for 1h, but the independent form lost its activity under the same conditions. Furthermore, 50% inhibition of the dependent enzyme activity, but only 10% inhibition of the independent enzyme activity, was observed with 0.1mM adenosine 3':5'-monophosphate (cAMP) when 1μM cGMP was employed as a substrate.View full abstractDownload PDF (811K) -
Kayoko NAKAMURA, Shizuo WATANABE1978 Volume 83 Issue 5 Pages 1459-1470
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin-like protein was obtained from E. coli by extraction with a sucrose solution and by precipitation with rabbit skeletal actin. The preparation of E. coli myosin-like protein looked very similar, in the sodium dodecyl sulfate-gel electrophoretic pattern, to that of rabbit skeletal myosin. The myosin-like protein was able to reversibly bind to rabbit actin. It had the activities of EDTA-, Ca-, and Mg-ATPases. The product in the EDTA-ATPase reaction catalyzed by the myosin-like protein was identified as ADP by ion exchange chromatography. The Mg-ATPase activity of E. coli myosin-like protein was activated by either rabbit actin or E. coli actin-like protein though the activation was much stronger by the latter. However, the myosin-like protein did not exhibit superprecipitation either with rabbit actin or with E. coli actin-like protein.
Actin-like protein was also obtained from E. coli by essentially the same procedures as those described for preparation of rabbit skeletal actin. E. coli actin-like protein was capable of activating Mg-ATPase of rabbit myosin, and also of superprecipitation with rabbit myosin. Extraction from both the whole cells and the membrane fraction of E. coli strongly suggested that the myosin-like protein and the actin-like protein are both localized in the membrane fraction rather than in the cytoplasmic fraction.View full abstractDownload PDF (2568K) -
Yukio IKEHARA, Kenji MANSHO, Keikichi TAKAHASHI, Keitaro KATO1978 Volume 83 Issue 5 Pages 1471-1483
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotein, was homogeneous in poly-acrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140, 000. The addition of β-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72, 000. Isoelectric focusing revealed that the pI of this enzyme is 4.7.
The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5-10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (Km=0.3mM) was most rapidly hydrolyzed. Vmax values of other substrates relative to that of p-nitrophenylphosphate were as follows; β-glycerophosphate, 76%; 5'-TMP, 82%; 5'-AMP, 62%; 5'-IMP, 43%; glucose-6-phosphate, 39%; ADP, 36%; and ATP, 15%.
More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55° C for 30min, or exposed either to 10mM β-mercaptoethanol for 10min, to 3M urea for 30min, or to an acidic pH below pH 5.0 for 2h. Of the effects by divalent cations, Mg2+ activated the enzyme by 20%, whereas Zn2+ strongly inhibited it by 95% at 0.5mM. EDTA at higher than 1mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1mM was reversible by the addition of divalent cations, particularly by Mg2+. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.View full abstractDownload PDF (2063K) -
Takachika AZUMA, Osamu KOBAYASHI, Yuji GOTO, Kozo HAMAGUCHI1978 Volume 83 Issue 5 Pages 1485-1492
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe circular dichroic (CD) spectra of a type λ Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type λ protein (Nag) were measured under various conditions. In the pH region from 5. 5 to 7. 5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VLdid change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4×104M-1 at pH 7. 5 and 25°C, which was smaller than that for VL (1×105M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains.View full abstractDownload PDF (585K) -
New Cross-Linking Reagent for Enzyme Labelling and a Preparation Method for Antiserum to ViomycinTsunehiro KITAGAWA, Takafumi FUJITAKE, Hyozo TANIYAMA, Tadaomi AIKAWA1978 Volume 83 Issue 5 Pages 1493-1501
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new cross-linking reagent of the hetero-bisfunctional type, a N-(m-maleimidobenzoyloxy)-succinimide (MBS) was prepared and used for enzyme labelling of viomycin under mild aqueous conditions by a two-step process. In the first step a maleimide residue was selectively introduced onto the N1-amino group of viomycin with a limited amount of MBS. The second step consisted of thioether formation between the maleimide residue and free thiol groups of β-D-galactosidase. An antiserum to viomycin was raised in rabbit by immunization with a viomycin-BSA conjugate. The conjugate was prepared by protecting the N6-amino group of viomycin with an acetyl group and succinylating the N1-amino group, activating the carboxyl group by a mixed anhydride method and coupling it with the amino groups of bovine serum albumin (BSA). The specificity of the antiserum was proved by an enzyme immunoassay based on the competition between viomycin and its enzyme conjugate toward diluted solutions of the antiserum. By use of the viomycin-enzyme conjugate and the antiserum to viomycin, enzyme immunoassay of viomycin was successfully performed by the competitive binding procedure with the double-antibody method, and 0.1 to 4ng of the antibiotic could be detected.View full abstractDownload PDF (621K) -
A Kinetic Discrimination of States of Tryptophan Residues Using N-BromosuccinimideHaruko FUJIMORI, Masatake OHNISHI, Keitaro HIROMI1978 Volume 83 Issue 5 Pages 1503-1510
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour tryptophan residues of saccharifying α-amylase from B. subtilis out of eleven in total are reactive towards N-bromosuccinimide (NBS), suggesting that they are on the surface of the enzyme. This is consistent with the results of solvent perturbation difference spectrophotometry with ethylene glycol.
One of four tryptophan residues was clearly distinguished from the other three in reactivity with NBS by the stopped-flow method. This most reactive tryptophan residue was not protected from modification by substrates or analogs, indicating that the tryptophan is not located in the substrate binding site.
One of the other three tryptophan residues, probably the second most reactive one, is considered to be related in some way to the glycosyl transfer in the reaction of the enzyme with maltose as a substrate.View full abstractDownload PDF (1794K) -
Akio MATSUKAGE, Taijo TAKAHASHI, Chikao NAKAYAMA, Mineo SANEYOSHI1978 Volume 83 Issue 5 Pages 1511-1515
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis is the first report dealing with the effect of 1-β-D-arabinofuranosylthymine 5' -triphosphate (araTTP), synthesized by a new method, on eukaryotic DNA polymerase [EC 2. 7. 7. 7]. AraTTP was tested for the inhibition of DNA synthesis in vitro using highly purified mouse myeloma DNA polymerase α in comparison with 1-β-D-arabinofuranosylcytosine 5'-triphosphate (araCTP). AraTTP was found to inhibit competitively the incorporation of [3H]dTTP into DNA and non-competitively the incorporation of [3H]dCTP, while the mode of the inhibition by araCTP was non-competitive with respect to dTTP and competitive with respect to dCTP. Neither araTTP nor araCTP was utilized as a substrate in place of dTTP or dCTP in DNA synthesis by DNA polymerase α.View full abstractDownload PDF (308K) -
Takao TAKI, Yoshio HIRABAYASHI, Yoshiko SUZUKI, Makoto MATSUMOTO, Kiyo ...1978 Volume 83 Issue 5 Pages 1517-1520
Published: May 25, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycolipid compositions of purified plasma membranes from rat ascites hepatomas, two island-forming cell-lines and two cell-lines of the free-type, and normal rat liver were compared. Ceramide monohexoside (CMH), ceramide dihexoside (CDH), and hematoside (GM3) were found in normal rat liver cell membranes. The island-type hepatomas contained ceramide trihexoside (CTH) and globoside besides CMH, CDH, and GM3. The free-type of hepatomas were characterized by the presence of asialo-type gangliosides but not GM3. Blood group H active fucolipid was a major glycolipid in the free-type of ascites hepatoma cell (AH 7974 F). The increase of glycolipid content in cell membranes seemed to be accompanied with a decrease of cell adhesiveness.View full abstractDownload PDF (1478K)
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