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Volume 93 (1983)
- Issue 6 Pages 1463-
- Issue 5 Pages 1221-
- Issue 4 Pages 943-
- Issue 3 Pages 669-
- Issue 2 Pages 323-
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Volume 93, Issue 6
Displaying 1-32 of 32 articles from this issue
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Koichi SUZUKI, Shoichi ISHIURA1983 Volume 93 Issue 6 Pages 1463-1471
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of μCANP, which is active at μM Ca2+, was also examined for comparison.
Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 μM. The structure of μCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of μCANP indicated that the structure of μCANP was looser or SH groups were more exposed than in the case of mCANP.
A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.View full abstractDownload PDF (692K) -
Michael E. BREIMER, Gunnar C. HANSSON, Karl-Anders KARLSSON, Hakon LEF ...1983 Volume 93 Issue 6 Pages 1473-1485
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcidic glycolipids (gangliosides and glycolipid sulphates) were purified from nonglycolipid contaminants by silicic acid chromatography of their acetylated derivatives. Acetylation converted gangliosides into non-polar neutral and weakly acidic derivatives. These were separated from acetylated sulphate-containing glycolipids by chromatography on DEAF-cellulose. The fractions thus isolated from small intestine of 7 different animals were analyzed by thin-layer chromatography. This revealed a considerable species-related variation of both gangliosides and sulpho-glycolipids. Mass spectrometry demonstrated a novel ganglioside in guinea-pig small intestine and a novel sulphoglycolipid in mouse small intestine.View full abstractDownload PDF (4640K) -
Yukihiro FUSAUCHI, Koichi IWAI1983 Volume 93 Issue 6 Pages 1487-1497
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe H2A histone of the protozoan Tetrahymena pyriformis was isolated by Bio-Gel P-10 chromatography from the H2A+H3 fraction obtained on a large scale, as described previously [Nomoto, M. & Iwai, K. (1982) J. Biochem. 91, 719-723], and further purified by Sephadex G-100 chromatography. The purified H2A was shown to comprise approximately equimolar amounts of two variants, H2A (1) and H2A (2), differing in molecular weight. The H2A mixture was fragmented with cyanogen bromide, yielding one N-terminal fragment (101 residues), one middle fragment (17 residues), and two C-terminal fragments (19 and 14 residues). The N-terminal fragment, whose N-terminal was blocked, was sequenced by overlapping its tryptic peptides with the peptides derived from the fragment with three proteases and the tryptic peptides from citraconylated intact H2A. One of the citraconylated tryptic peptides showed the arrangement of the N-terminal, middle, and C-terminal fragments; the latter two fragments were directly sequenced by Edman degradation. Thus, the total sequences of H2A (1) and H2A (2) were completely determined; the two variants differ in the total residue number, 137 or 132, the molecular weight in the unmodified form, 14, 654 or 14, 126, His or Asn at residue 40, Ser or Thr at residue 124, and the C-terminal sequence at residues 128-137 or 128-132, respectively. These sequences are compared with the known H2A sequences, and the implications for the structure and function relationship of this histone species are discussed.View full abstractDownload PDF (755K) -
Ikuko SAGAMI, Minro WATANABE1983 Volume 93 Issue 6 Pages 1499-1508
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA major form of pulmonary cytochrome P-450 (pulmonary P-450 MC) was purified approximately 313 fold from lung microsomes of 3-methylcholanthrene (MC)-treated rats. The purified preparation contained 12.5 nmol of cytochrome P-450 per mg protein and was essentially free from NADPH-cytochrome c-reductase, NADH-cytochrome b5-reductase, and cytochrome b5. By SDS-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450 MC was estimated to be 54, 000, which is smaller than that of a major form of hepatic cytochrome P-450 (hepatic P-450 MC) purified from MC-treated rats. The peptide patterns of the purified pulmonary P-450 MC were apparently different from those of hepatic P-450 MC on partial proteolysis with either S. aureus V 8 protease or papain, indicating that the primary structure of pulmonary P-450 MC is different from that of hepatic P-450 MC. In reconstituted systems, pulmonary P-450MC efficiently catalyzed benzo (a) pyrene hydroxylation and ethoxycoumarin O-deethylation, but showed a low activity for benzphetamine N-demethylation. Pulmonary P-450 MC formed a single precipitation line with the antibody prepared against hepatic P-450 MC in Ouchterlony double diffusion analysis. The pulmonary and hepatic P-450 MC activities of benzo (a) pyrene hydroxylation and ethoxycoumarin O-deethylation were inhibited in the same manner by the antibody against hepatic P-450 MC.These results suggest that pulmonary P-450 MC is immunologically very similar to hepatic P-450 MC.View full abstractDownload PDF (2267K) -
Hideo YAMAGATA1983 Volume 93 Issue 6 Pages 1509-1515
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEscherichia coli mutant Y815 accumulates the precursor of lipoprotein (prolipoprotein) in its envelope. The accumulated prolipoprotein could be chased to mature lipoprotein at 30°C but not at 60°C (Yamagata, H., Ippolito, C., Inukai, M., & Inouye, M. (1982) J. Bacteriol. 152, 1163). When the envelope fraction prepared from the mutant was mixed with the envelope fraction prepared from wild-type E. coli cells and incubated at 60°C in the presence of Triton X-100, the prolipoprotein in the mutant envelope fraction was cleaved rapidly to mature lipoprotein. The cleavage was dependent on the addition of wild-type envelope fraction and Triton X-100 to the reaction mixture. This indicated that the prolipoprotein accumulated in the mutant envelope is a good substrate for the signal peptidase which cleaves the signal peptide from the prolipoprotein, and hence the accumulation of prolipoprotein was due to lack of the signal peptidase in the mutant. The optimum concentration of Triton X-100 for the cleavage of the prolipoprotein in the above in vitro system was 0.05 to 0.1% (v/v) at a wild-type envelope concentration of 0.35 mg protein/ml. Prolipoprotein accumulated in wild-type cells on treatment with globomycin, a specific inhibitor of the signal peptidase, was also cleaved to mature lipoprotein under the same conditions. Triton X-100 was shown to solubilize the signal peptidase from the envelope fraction. The cleavage of the prolipoprotein was rapid and complete in the in vitro system described here, which provides an efficient and convenient assay system for the solubilized signal peptidase for prolipoprotein.View full abstractDownload PDF (2946K) -
Tsuneo IMANAKA, Kimiko AMANUMA-MUTO, Shoji OHKUMA, Tatsuya TAKANO1983 Volume 93 Issue 6 Pages 1517-1521
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of phospholipids on lysosomal acid lipase purified from rabbit liver were studied. Non-ionic phospholipids such as phosphatidylethanolamine and phosphatidylcholine increased the enzyme activity. However, anionic phospholipids such as phosphatidylserine, phosphatidylinositol, and cardiolipin were ineffective. The acyl chain length and the degree of unsaturation of synthetic phospholipids were also related to the enzyme activity. Among a series of saturated phosphatidylcholines with different aryl chain lengths, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine was the most effective. Among a series of unsaturated phosphatidylcholines, the enzyme activity increased in parallel with the number of double bonds. The role of lysosomal acid lipase in relation to phospholipids is discussed.View full abstractDownload PDF (440K) -
Kazuhisa SEKIMIZU, Masami HORIKOSHI, Hideki OKAJIMA, Shunji NATORI1983 Volume 93 Issue 6 Pages 1523-1530
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA procedure was developed for large scale purification of RNA polymerase II from Ehrlich ascites tumor cells. About 2mg of purified enzyme was obtained from 800g of wet cells. Ten subunits were identified which behaved corresponding to the enzyme activity both on DEAE-Sephadex chromatography and on glycerol density gradient centrifugation. Analysis of tryptic peptides of each subunit showed that these subunits were independent proteins and not structurally related to each other.View full abstractDownload PDF (2090K) -
Takeyori SAHEKI, Mariko SASE, Kyoko NAKANO, Fumio AZUMA, Tsunehiko KAT ...1983 Volume 93 Issue 6 Pages 1531-1537
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an attempt to clarify the pathogenesis of citrullinemia, which is caused by argi. ninosuccinate synthetase (ASS) [EC 6. 3. 4. 5] deficiency and is observed in extraordinarily high incidence in Japan, some properties of ASS purified to homogeneity from human liver were compared with those of ASS purified from rat liver.
The molecular weights of the subunit and native form of purified human liver ASS were estimated to be 45, 000 and 185, 000, respectively, which are the same as those of rat liver ASS. Human liver ASS migrated as a single band on polyacrylamide gel electrophoresis at pH 8.9 but showed multiple bands at pH 6.6 and on isoelectrofocusing in thin layer polyacrylamide gel, as did rat liver ASS. There was almost no difference in the apparent Km values for three substrates between human and rat liver ASSs. Both enzymes showed negative cooperativity in respect to ATP. Rat liver ASS also showed negative cooperativity in respect to one amino acid substrate with the other amino acid substrate at unsaturated concentrations, as reported for bovine liver ASS, while human liver ASS showed only slight cooperativity effects.
Immunological analyses also showed a high degree of similarity between the enzymes. The amino acid composition of human liver ASS was very similar to that of rat liver ASS. Differences in the amounts of amino acid residues per subunit between human and rat liver ASSs were within 10% except for serine, phenylalanine, and arginine. The peptide patterns on SDS-polyacrylamide gel electrophoresis after digestion of the enzymes by trypsin were almost indistinguishable from each other. These results suggest that the primary structures of the human and rat liver enzymes are very similar.View full abstractDownload PDF (1719K) -
Fumio SHISHIKURA, Koichi SEKIGUCHI1983 Volume 93 Issue 6 Pages 1539-1546
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAgglutinins from Tachypleus (Tachypleus tridentatus, the Japanese horseshoe crab) hemolymph were isolated by affinity chromatography on BSM-coupled Sepharose 4 B. The agglutinins showed multiple species and were composed of eight heterogeneous subunits with molecular weights of 45, 000, 42, 000, 41, 000, 39, 000, 33, 000, 29, 000, 27, 000, and 22, 000 as determined by SDS-polyacrylamide gel electrophoresis. The affinity-isolated agglutinins were fractionated into four groups by gel filtration on a Fractogel TSK (Toyopearl) HW-65 column, and these were designated as Tachypleus tridentatus agglutinin (TTA)-I, -II, -III, and -IV in the order of elution. These agglutinins were demonstrated to be heterogeneous as judged by their specificity towards horse erythrocytes, subunit structures, and immunological properties. TTA-III showed a potent agglutination activity towards horse erythrocytes and was further purified by gel filtration on a Cellulofine GC-700 column. The purified TTA-III is a highly purified (46, 000-fold) protein composed of homogeneous subunits (Mr, 42, 000) as judged by SDS-polyacrylamide gel electrophoresis and immunological analysis.View full abstractDownload PDF (3328K) -
Osamu OHARA, Sho TAKAHASHI, Tatsuo OOI1983 Volume 93 Issue 6 Pages 1547-1556
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have recently reported that actin modified with dimethyl suberimidate takes a filamentous form even under depolymerizing conditions, and this phenomenon is accounted for by the conformational fixation caused by the introduction of an intramolecular cross-link (Ohara, O., Takahashi, S., Ooi, T., & Fujiyoshi, Y. (1982) J. Biochem. 91, 1999-2012). The suberimidate-treated actin (SA) is not immediately depolymerized by deoxyribonuclease I (DNase I) but is depolymerized after incubation for one day, i.e., depolymerization is much slower than that for intact Factin. The results on circular dichroic spectra of a mixture of SA and DNase I suggest that DNase I flips the conformation of SA into a G-actin-like state from the F-actin-like one when a tight SA-DNase I complex is formed. The suberimidate cross-link introduced in an SA molecule does not completely prevent the conformational change from the F-state to the G-state but stabilizes the actin conformation very greatly in the F-state.View full abstractDownload PDF (1543K) -
Fumio TASHIRO, Akihiro SHIBATA, Natsue NISHIMURA, Yoshio UENO1983 Volume 93 Issue 6 Pages 1557-1566
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSZearalenone is known to be reduced by rat liver preparations to a more active estrogenic metabolite, α-zearalenol. To elucidate the enzymatic feature of zearalenone reductase, we worked out a simple assay procedure for this enzyme using [3H]-zearalenone and thin layer chromatography (TLC).
The NAD (P) H-dependent zearalenone reductase localized in the microsomes was most active at pH 4-4.5, while the reductase localized in the cytosol was active at neutral pH. The former enzyme reduced zearalenone only to α-zearalenol and the latter reduced both α-zearalenol and β-zearalenol.
The microsomal enzyme was solubilized with Triton X-100 and purified by DEAE-Sephadex, hydroxyapatite and Sepharose 4 B column chromatographies. The partially purified enzyme showed the following properties. a) The molecular weight of the enzyme was estimated to be about 230, 000 by Sepharose 4 B chromatography. b) The apparent Km value of the enzyme was 1.0×10-5M for zearalenone at an NADPH concentration of 1mM. c) The enzyme activity was inhibited by a high concentration of KCl but not by 1mM Co2+, Mn2+, Zn2+, EDTA, or 20mM N-ethylmaleimide.View full abstractDownload PDF (756K) -
Shigeyuki TERASHITA, Toyoki KATO, Hidemi SATO1983 Volume 93 Issue 6 Pages 1567-1574
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS21 S Dynein ATPase [EC 3. 6. 1. 3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [γ-32P]ATP at various concentrations, 5mM divalent cations, and 20mM imidazole at pH 7.0 and 0°C. The following results were obtained. 1. 21 S Dynein had a latent ATPase activity of about 0.63 pmol P1/(mg•min) in 1mM ATP, 100mM KCI, 4mM MgSO4, 0.5mM EDTA, and 30 mM Tris-HC1 at pH 8.0 and 25°C. Its exposure to 0.1% Triton X-100 for 5min at min) 25°C induced an increase in the ATPase activity to about 3.75 μmol P1/(mg) and treatment at 40°C for 5min also induced a similar activation. 2. The doublereciprocal plot for the ATPase activity of dynein activated by the treatment at 40°C consisted of two straight lines, while that of nonactivated 21 S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21 S dynein showed substrate inhibition at ATP concentrations above 0.1mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of P, liberation. The apparent P1-burst size was 1.0 mol/(106g protein) and the true size was calculated to be 1.6 mol/1, 250 K after correcting for the effect of P1 liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21 S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of P1 liberation of 1.4 mol/(106g protein) in the presence of 54% (v/v) glycerol.View full abstractDownload PDF (565K) -
Shigeyuki TERASHITA, Toyoki KATO, Hidemi SATO, Yuji TONOMURA1983 Volume 93 Issue 6 Pages 1575-1581
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amounts of ATP and ADP bound to 21S dynein during the ATPase reaction were measured in the presence of 2.83mg/ml 21S dynein, 2mM PEP, 4mg/ml PK, 0.1M KCl, 5mM MgCl2, 1mM DTT, 0.1mM PMSF, 50% [2-3 H]glycerol, and 20mM imidazole at pH 7.0 and 0°C. The maximum amounts of ATP and ADP bound to 21 S dynein were 0.29 and 0.55 mol/(106g protein), respectively. The dissociation constants of ATP for the ATP and ADP binding (4 μM) were almost equal to the Km value (3.7 μM) of dynein-ATPase in the steady state. The amount of bound ADP during the initial phase showed an overshoot, which reached 0.6-0.8 mol/106g protein at 5s, then decreased to the steady state level within 20s. Furthermore,
the rate of TCA-P1 liberation during the initial 5s was 6 times the steady-state rate. The apparent P, -burst size, estimated by extrapolating the steady-state P1 liberation to zero time, was 1.33 mol/(106g protein). The true P1-burst size was calculated to be 1.56 mol/(106g protein) by correcting for the effect of P1 liberation at steady state.
All these findings could be explained quantitatively by the following reaction scheme for 21 S dynein ATPase in the presence of glycerol:
_??_
where K1=25.5 μm, and k2, k3, and k4 were 0.39, 0.21, and 0.11 s-1, respectively.View full abstractDownload PDF (594K) -
Saori TAKAHASHI, Tamiko OHSAWA, Retsu MIURA, Yoshihiro MIYAKE1983 Volume 93 Issue 6 Pages 1583-1594
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRenin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4 B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42, 000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37°C between pH 5.0 and 9.0 or on storage for 4 weeks at 4°C or -80°C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nivt, assuming that the stoichi-ometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparant molecular weight of the reconstituted specimen was estimated to be 60, 000 by gel filtration on Ultrogel AcA 44.View full abstractDownload PDF (4104K) -
Etsuko YASUGI, Takeshi KASAMA, Hideya KOJIMA, Tamio YAMAKAWA1983 Volume 93 Issue 6 Pages 1595-1599
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEster cerebrosides isolated from whale brain were demonstrated to be a mixture of 6-O-acyl, 2-O-acyl, 4-O-acyl, and 3-O-acyl galactosyl ceramides in that order of content (Yasugi, E., et al. (1982) J. Biochem. 91, 1121-1127). However, the existence of 2-O-acyl and 4-O-acyl galactosyl ceramides has not been reported for other mammalian brains. In the present work, ester cerebrosides isolated from bovine and human brains were examined in order to determine whether the above-mentioned substitution is only present in whale brain or also present in other mammalian brains. For determining the substituted positions of the acyl group on the galactose moiety, free hydroxyl groups of ester cerebrosides were protected with dihydropyran, deacylated by mild alkali treatment, and then subjected to permethylation. Finally, the methylated galactitol acetates obtained by hydrolysis and reduction were analyzed by gas chromatography and gas chromatography/mass spectrometry. By these procedures, ester cerebrosides obtained from bovine and human brains were demonstrated to be not only 6-O-acyl and 3-O-acyl galactosyl ceramides but also 2-O-acyl and 4-O-acyl galactosyl ceramides similar to those of whale brain.View full abstractDownload PDF (1023K) -
Junshi SAKAMOTO, Yuji TONOMURA1983 Volume 93 Issue 6 Pages 1601-1614
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe found that ATP is synthesized by mitochondria) soluble F1-ATPase from medium ADP and Pi in the presence of dimethylsulfoxide (DMSO), with the amount synthesized increasing with the DMSO concentration to a maximum at 30% (w/v) DMSO. In the presence of 35% (w/v) DMSO, ADP was scarcely converted into AMP and much more ATP was formed than AMP. The pH dependence curve of ATP synthesis was bell-shaped with the optimum at 6.7. The amount of synthesized ATP measured after stopping the reaction with triehloroacetic acid was almost equal to that measured after stopping the reaction with sodium dodecyl sulfate or with ethanol. Therefore, we measured the amount of ATP synthesized by F1 from ADP and P1 in the presence of 4.2mM Mg2+ and 35% (w/v) DMSO at pH 6.7 and 30°C after stopping the reaction with triehloroacetic acid. The following results were obtained.
1. The rate and extent of [α-32P] ATP synthesis from [α-32P] ADP and P, were equal to those of [γ-32P]ATP synthesis from ADP and 32P1.
2. The ATP synthesized was inaccessible to hexokinase, and its amount was proportional to that of F, The ATP synthesis was inhibited by sodium azide, but not by 7-chloro-4-nitro-2, 1, 3-benzoxadiazole, or by N, N'-dicyclohexylcarbodiimide.
3. No nucleoside 5'-triphosphate was synthesized by Fl, when GDP, IDP, CDP, or UDP was used as a substrate.
4. Both the dependence on ADP concentration of the amount of ATP formed in the presence of a sufficient concentration of Pi and the dependence on P1 concentration of the amount in the presence of a sufficient concentration of ADP were given by the following equation:
[ATP formed]=[ATP formed]max/(1+KADP/[ADP] or Kp/[P1])
where KADP=3 μM, Kp=0.55mM, and [ATP formed]max=0.4-0.6 mol/mol F1.
5. When the reaction mixture was diluted with the buffer solution after the ATPsynthesis reaction had reached equilibrium, the amount of synthesized ATP decreased monophasically at a higher rate than that of ATP formation. When the pH of the reaction mixture was rapidly increased from 6.9 to 8.0, about half of the synthesized ATP disappeared very rapidly, while the remainder decreased rather slowly.
6. All these findings can be explained by the following reaction scheme in which the catalytic sites in F1 for ATP synthesis are assumed to function independently:
_??_
where the brackets indicate tight binding.
7. However, the dependence on P1 concentration of the initial rate of ATP synthesis, vf, in the presence of a sufficient amount of ADP was given by the equation, vf=Vf•max/[1+(Kp'/[P1])2]. Furthermore, when AMPPNP was added to the reaction mixture, 60-70% of the formed ATP disappeared very rapidly and the remainder decreased very slowly. These two findings suggest cooperativity between catalytic or nucleotide-binding sites of F1 during the ATP-synthesis reaction.View full abstractDownload PDF (1696K) -
Ken EBISAWA1983 Volume 93 Issue 6 Pages 1615-1619
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSContractile and regulatory proteins were prepared from bovine aortic intima, and actin from bovine stomach smooth and rabbit skeletal muscles. In the desensitized and reconstituted actomyosin system, the superprecipitation activity was measured by the turbidity method. Superprecipitation of each system was not exhibited even in the presence of Ca ions, but was observable only in the presence of tropomyosin and Ca ions, while 20, 000-dalton light chain of myosin remained dephosphorylated during the reaction. Addition of tropomyosin to the reconstituted acto-myosin digest system (trypsin-digested myosin was devoid of 20, 000-dalton light chain) also restored the Ca2+-sensitivity. These results indicate that the phosphorylation of myosin light chain is not a crucial step in the contraction of aortic intima smooth muscle. For full activation of the actin-myosin-ATP interaction, additional factors other than the myosin light chain kinase are required, although some contribution of the kinase to the full activation cannot be ruled out.View full abstractDownload PDF (1704K) -
Yasuo SUZUKI, Takashi SUZUKI, Makoto MATSUMOTO1983 Volume 93 Issue 6 Pages 1621-1633
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSialoglycoprotein which exhibits inhibitory activity for hemagglutination by Hemagglutinating Virus of Japan (HVJ, Sendai virus) was isolated from the membrane of bovine erythrocytes. Purification steps for this sialoglycoprotein included extraction with lithium diiodosalicylate, phenol partition, precipitation with ethanol, and chromatography on a phosphocellulose column and an SDS-Sepharose CL-4B column. Purified sialoglycoprotein (GP-2) has high specific activity for inhibiting the hemagglutination with HVJ, and a lesser activity for that with Newcastle disease virus, but it does not inhibit the hemagglutination by influenza A virus. Inhibitory activity of GP-2 on hemagglutination by HVJ is 2, 500-fold higher than that of fetuin. Liposomes containing a 10, 000-fold larger amount of ganglioside mixture of bovine erythrocytes and those containing a 5, 000-fold larger amount of each ganglioside of bovine erythrocytes, N-glycolylneuraminosyl-lactosyl ceramide, sialosyllacto-N-neotetraosyl-and sialosyl-lacto-N-norhexaosyl ceramide, had no inhibitory activity toward hemagglutination with HVJ.
GP-2 (mol. wt. 250 K daltons) behaved homogeneously in SDS-polyacrylamidegel electrophoresis. It contained 70% carbohydrate and 30% protein, by weight. N-Acetylgalactosamine, N-acetylglucosamine, galactose, sialic acid (N-glycolyl-neuraminic acid, 96%; N-acetylneuraminic acid, 4%) were identified as carbohydrate components, in molar ratios of 1.0:4.0:5.2:2.9. All the oligosaccharides of GP-2 appeared to be linked to polypeptide chains by alkali-labile O-glycosidic linkages. Sialidase treatment of GP-2 and conversion of sialic acid residue of the glycoprotein to C8 and C7 analogues resulted in the loss of the inhibitory activity on hemagglutination by HVJ. Oligosaccharides isolated by gel filtration after treatment of GP-2 with alkaline borohydride had also lost the ability to inhibit the hemagglutination by HVJ.
The above results indicate that isolated sialoglycoprotein is the endogenous receptor in bovine erythrocyte membrane specific to HVJ, and the hydroxy group linked to the 9-carbon atom of sialic acid and probably also the hydrophobic protein moiety are important for the recognition of HVJ attachment.View full abstractDownload PDF (2243K) -
Koichi ISHII, Hiroshi SAGAMI, Kyozo OGURA1983 Volume 93 Issue 6 Pages 1635-1639
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe microsomes from pig liver contained farnesyl pyrophosphate synthetase and it was solubilized with Triton X-100. The microsomal enzyme had a pH optimum of 6.5-7.0 and required Mg2+, or Mn2+ for maximum activity. Dimethylallyl-transferring activity of the enzyme was much lower compared with the geranyl-transferring activity. In the presence of Triton X-100, the geranyl-transferring activity was about two-fold activated whereas the dimethylallyl-transferring activity was almost the same.View full abstractDownload PDF (388K) -
Hiroaki YOSHIDA, Shigeyuki FUKUI, Ikuo FUNAKOSHI, Ikuo YAMASHINA1983 Volume 93 Issue 6 Pages 1641-1648
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe membrane-bound enzyme responsible for the breakdown of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) has been purified 1, 900-fold from detergent-solubilized human placenta, using chromatographies on Con A-Sepharose, Blue Sepharose, AMP-Agarose, and Sepharose CL-6B, and sucrose density gradient centrifugation. The enzyme required Mg2+ and showed the optimum activity at pH 9.4. The preparation was free of alkaline phosphatase [EC 3. 1. 3. 1], phosphodiesterase [EC 3. 1. 4. 1], and 5'-nucleotidase [EC 3. 1. 3. 5] activities, which enabled investigation of the substrate specificity and kinetic properties of the enzyme without interference by secondary reactions due to the above activities.
The enzyme cleaved the pyrophosphate linkages of NAD and various sugar nucleotides and the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate (PNTP), as well as the phosphosulfate linkages of PAPS and its biosynthetic precursor, adenosine 5'-phosphosulfate (APS), with apparent Km values of 0.12-0.33mM. Relative activities towards PNTP and PAPS did not change during the purification procedures starting from the homogenate. This, together with the data of kinetic studies using two substrates simultaneously, led us to conclude that the activities towards all the substrates tested were due to one and the same nucleotide pyrophosphatase.View full abstractDownload PDF (655K) -
Noboru YAMADA, Kiyomi TSUKAGOSHI, Masaru KAWAMURA, Koscak MARUYAMA1983 Volume 93 Issue 6 Pages 1649-1653
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDepartment of Biology, Faculty of Science, Chiba University Amorphous aggregates of actin of various sizes were observed under an electron microscope, when actin polymerization was inhibited or retarded by EDTA or by low salt concentration. It was first thought that these aggregates could be intermediate complexes of actin in the process of polymerization. These aggregates were, however, present in usual G-actin solution as well as column-chromatographed or highly clarified samples. In the samples prefixed with glutaraldehyde, the aggregates were also observed. Moreover, similar aggregates were seen in denatured actin samples. Hence it is concluded that these aggregates of actin are artefact products due to the treatment with uranyl acetate used for negative staining.View full abstractDownload PDF (1257K) -
Yuji SHIMADA, Yoshio TOMINAGA, Mieko IWAI, Yoshio TSUJISAKA1983 Volume 93 Issue 6 Pages 1655-1660
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRhizopus (Rh.) delemar (ATCC 34612) C-lipase was found to exhibit a slight activity towards water-soluble esters. The hydrolytic reaction of this lipase on α-naphthyl acetate was competitively inhibited by the presence of olive oil or Tween 80. This finding showed that both substrates, insoluble triglyceride and water-soluble ester, were hydrolyzed at the same site on the enzyme.
The activities on water-soluble esters (α-naphthyl acetate, β-naphthyl acetate, methyl acetylsalicylate and Tween 80) increased on binding of lipase with phosphatidylcholine (PC), although the activity on olive oil did not change. The increase in activity on water-soluble esters was due to the increase in the Vmax for its hydrolysis. It appears that local structural change of the catalytic site on lipase occurred on binding of PC to the lipase molecule and resulted in an increase in the activity on water-soluble esters. The temperature dependence of the hydrolysis of water-soluble esters demonstrated that the activation energy was lowered on binding of PC to the lipase molecule, and this resulted in an increase in the activity.View full abstractDownload PDF (459K) -
Michiyuki YAMADA, Midori MORI, Takashi SUGIMURA1983 Volume 93 Issue 6 Pages 1661-1668
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman myeloid leukemia HL-60 cells were grown either in suspension culture or in nude mice. The two types of myeloperoxidase, the small and the large type, in crude extracts of these cells were analyzed by sucrose density gradient centrifugation. The proportions of the small and the large myeloperoxidase varied markedly depending on the growth conditions of cells. In cells in culture, the small myeloperoxidase amounted to 80% of the total myeloperoxidase, whereas in the solid tumors it amounted to only about 30% of the total. Both in cultured cells and solid tumors, 40% of the total myeloperoxidase was found in the soluble fraction and the rest in the granule fraction. However, adult and fetal blood granulocytes contained only the large myeloperoxidase, which was mainly recovered in the granule fraction. Antiserum prepared against purified large myeloperoxidase of HL-60 solid tumors reacted with the small myeloperoxidase as well as the large enzyme of HL-60 cells in culture. The antiserum also precipitated myeloperoxidase of adult and fetal blood granulocytes. An Ouchterlony double immunodiffusion test also revealed their immunological identity.View full abstractDownload PDF (1650K) -
Hirokazu KATAYAMA, Yasuo KITAGAWA, Etsuro SUGIMOTO1983 Volume 93 Issue 6 Pages 1669-1675
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn increase of rat liver glycerate kinase [ATP: D-glycerate 2-phosphotransferase EC 2. 7. 1. 31] on protein feeding, on starvation or during fetal and neonatal development was studied from the viewpoint of the role of glucagon as the mediator. On starvation and during fetal and neonatal development, glycerate kinase in the cytosolic and mitochondrial fractions increased approximately in the same ratio. Glucagon in plasma increased under these conditions and glucagon administration increased glycerate kinase in both intracellular fractions approximately in the same ratio. The above results suggested that glucagon functions as at least one of the mediators for these processes. In diabetic rats, only a slight increase of the enzyme in both intracellular fractions was observed. Protein feeding, however, strongly increased the mitochondrial enzyme. Glucagon failed to mediate this function of dietary protein for the mitochondrial enzyme.View full abstractDownload PDF (640K) -
Chiseko NODA, Fukiko SHINJYO, Toshikazu NAKAMURA, Akira ICHIHARA1983 Volume 93 Issue 6 Pages 1677-1684
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of glucagon, dexamethasone (Dex), and isoproterenol (Ip) on induction of serine dehydratase [EC 4. 2. 1. 13] in primary cultured hepatocytes were investigated. Both glucagon and Dex were necessary for induction of this enzyme. Activity started to increase after a 6-h lag period and reached a maximum 24 h after addition of the hormones. This lag period could be diminished by pretreating the cells with Dex alone for 12h and then adding glucagon. The reverse order of additions of hormones did not cause enzyme induction and the loss of induction was due to desensitization of response to glucagon at a step of cyclic AMP (cAMP) formation.
In the presence of Dex, glucagon induced serine dehydratase, whereas Ip did not. This was because glucagon could maintain a high level of cAMP in the cells for 6h, whereas Ip increased the cAMP level for only a short time, which was not sufficient to induce serine dehydratase. Thus, when the cells were treated with Ip plus 1-methyl-3-isobutylxanthine (MIX), the intracellular cAMP concentration remained high for over 6h and serine dehydratase was induced to the maximum level. Similarly, the active state of cAMP-dependent protein kinase [EC 2. 7. 1. 37] was maintained for over 6 h when the cells were treated with either glucagon or Ip plus MIX, but not with Ip alone. When cells were treated with various concentrations of glucagon, the intracellular concentration of cAMP at 3 h correlated well with the induced level of enzyme activity at 24 h after hormone treatment.
These results indicate that for induction of serine dehydratase, the action of glucocorticoid is a prerequisite, and that it is followed by a cAMP-dependent action of glucagon. Maintenance of a high cAMP level for a prolonged period is necessary for induction of this enzyme, unlike for the inductions of other enzymes such as tryptophan oxygenase [EC 1. 13. 11. 11] and tyrosine aminotransferase [EC 2. 6. 1. 5].View full abstractDownload PDF (693K) -
Naoko SAKIHAMA, Kazunori NIIMI, Masaji NAKAMURA, Masateru SHIN1983 Volume 93 Issue 6 Pages 1685-1690
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpinach ferredoxin was trinitrophenylated by reaction with 2, 4, 6-trinitrobenzenesttlfonate. Four amino groups in the ferredoxin could be modified of the total of five amino groups. The trinitrophenylated ferredoxin formed a complex with ferredoxin-NADP+ reductase just as native ferredoxin did. The modified ferredoxin also retained the activity of electron transport in the cytochrome c photoreduction system of chloroplasts, but could neither donate electrons to ferredoxin-NADP+ reduetase in the NADP+ photoreduction system, nor accept electrons from the reductase in the NADPH-cytochrome c reduction system in vitro. Furthermore, it lost the inhibitory effect against the NADPH-diaphorase activity of the reductase. These results suggest that the complex formation of ferredoxin with ferredoxin-NADP- reductase is a phenomenon essentially independent of the function of electron transport between the two proteins.View full abstractDownload PDF (460K) -
Kazushige TAKAHASHI, Toshihide KOBAYASHI, Akiko YAMADA, Yutaka TANAKA, ...1983 Volume 93 Issue 6 Pages 1691-1699
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe shedding of acetylcholinesterase-enriched vesicles from erythrocytes of various species of animals occurred when cells were treated with C12:0PC. The response was observed shortly after a morphological change of erythrocytes without any accompanying detectable K+ leakage or hemolysis. The vesiculation was inhibited by the presence of serum albumin or by the incorporation of cholesterol into C12:0PC liposomes, indicating that the insertion of C12:0PC into the erythrocyte membrane causes the vesiculation. The ratio of C12:0PC to total phospholipid determined in vesicle fractions was almost the same as that observed in non-hemolyzed cell fractions. This finding suggests that the vesicles were not shed from portions of membranes rich in C12:0PC.
The vesicles showed similar characteristics to those generated by ATP depletion; their diameter is 150-200 nm and they are enriched with acetylcholinesterase activity. Erythrocytes became denser when they lost acetylcholinesterase activity on treatment with C12:0PC.View full abstractDownload PDF (1312K) -
Shoji ODANI, Takehiko KOIDE, Teruo ONO1983 Volume 93 Issue 6 Pages 1701-1704
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the carboxyl-terminal half of barley trypsin inhibitor was found to be significantly similar to the whole sequence of bovine pancreatic secretory trypsin inhibitor (Kazal). Kazal type inhibitors and related proteins are known for the extraordinary mode of divergence among animals, and the present observation extends this to a plant for the first time. The present observation together with our previous finding of sequence homology between barley trypsin inhibitor and wheat α-amylase inhibitor (Odani, S., Koide, T., & Ono, T. (1982) FEBS Lett. 141, 279-282) suggest an unusual evolutionary relationship between cereal enzyme inhibitors and animal proteinase inhibitors of the Kazal type.View full abstractDownload PDF (316K) -
Koshin MIHASHI, Hideyuki YOSHIMURA, Takuhiro NISHIO, Akira IKEGAMI, Ka ...1983 Volume 93 Issue 6 Pages 1705-1707
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy means of laser flash photolysis, the transient absorption anisotropy (TAA) of the triplet probe, 5-iodoacetamide-Eosin, labeling rabbit skeletal F-actin was measured in the 10-6-10-3 s time range. The TAA curve at 20°C showed a relatively slow decay phase covering several hundred microseconds and a large residual anisotropy (_??_0.1 at 2 ms). After analysis with Barkley & Zimm's formula, it was concluded that the TAA of Eosin-F-actin can be approximated by the anisotropy decay due to torsional motion of F-actin.View full abstractDownload PDF (274K) -
Yukiko YAMASHITA, Hiroyuki SUGIYAMA1983 Volume 93 Issue 6 Pages 1709-1712
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree methods were compared for detecting monoclonal antibodies against Narke japonica acetylcholine receptors as to the sensitivity of the detection and the convenience of operation. One of the methods involves the use of a microplate lid with 96 attached plugs that are immersed into opposite wells, and requires very simple operations. This method was found to be highly sensitive and could detect specific antibodies at levels of less than 150 pg/ml in the culture medium of hybridoma cells.View full abstractDownload PDF (287K) -
Hiroshi NOJIMA1983 Volume 93 Issue 6 Pages 1713-1716
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe published primary and secondary structures of small nuclear RNAs (snRNAs) (1-9) were reexamined and a new common model for their secondary structures was proposed. What is surprising is the resemblance of their secondary structures. U1, U2, U4, and U5 snRNAs share a common sequence of A(U)nG (n_??_3) at the single-stranded junction between two hairpins. U6 RNA does not have the A(U)nG sequence, however, all of its possible loops have common sequences with those of U1 RNA at exactly the same positions. Other remarkable common sequences are also noted and their possible biological significance is discussed.View full abstractDownload PDF (345K) -
Hiroh IKEZAWA, Toshikatsu NAKABAYASHI, Koichi SUZUKI, Masahiro NAKAJIM ...1983 Volume 93 Issue 6 Pages 1717-1719
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA phosphatidylinositol-specific phospholiase C was purified from the culture broth of Bacillus thuringiensis IAM 12077 to a homogeneous state as revealed by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 559 units/mg and recovery of the enzyme activity was 31%. Molecular and physiological properties of the purified enzyme, including molecular weight (22, 000), isoelectric point (p1=4.9) and its ectoenzyme-releasing activity, were studied in comparison with those other known enzymes of bacterial origin.View full abstractDownload PDF (938K)
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