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Volume 99 (1986)
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- Issue 5 Pages 1289-
- Issue 4 Pages 993-
- Issue 3 Pages 607-
- Issue 2 Pages 315-
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Volume 99, Issue 5
Displaying 1-33 of 33 articles from this issue
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Yasuyuki KITAGAWA, Susumu TSUNASAWA, Nobuo TANAKA, Yukiteru KATSUBE, F ...1986 Volume 99 Issue 5 Pages 1289-1298
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of Cu, Zn-superoxide dismutase (SOD) from spinach leaves has been determined on the basis of peptides obtained by cyanogen bromide (BrCN) cleavage and by enzymic hydrolyses with Achromobacter lyticus lysylendopeptidase, Staphylococcus aureus V8 protease, trypsin, and thermolysin. The spinach SOD consists of a total of 154 amino acid residues with alanine as the amino(N-)terminus and valine as the carboxy(C-)terminus. The present sequence, which has been established for the enzyme from a plant, is also highly homologous to those of the enzymes from other species. Especially, the residues essential for metal binding and enzyme activity have been extensively conserved among all of the Cu, Zn-SODs hitherto analyzed.View full abstractDownload PDF (699K) -
Sumio ISHIJIMA, Katsura IZUI, Hirohiko KATSUKI1986 Volume 99 Issue 5 Pages 1299-1310
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.View full abstractDownload PDF (1738K) -
Nobuo NAKANISHI, Kenichi OZAWA, Shozo YAMADA1986 Volume 99 Issue 5 Pages 1311-1315
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been difficult to determine exactly NADPH-specific dihydropteridine reductase [EC 1.6.99.10] in samples which also contain NADH-specific dihydropteridine reductase [EC 1.6.99.7], because the latter enzyme interferes with the activity measurement of the former. We have devised a method to measure selectively the NADPH-specific reductase in crude extracts of bovine, human and monkey livers by the single radial immunodiffusion method using specific antiserum against the enzyme. This method makes it possible to determine the enzyme amount in 5 μl of the 3-volume extracts of the livers. The amounts of NADPH-specific dihydropteridine reductase were calculated to be 0.252, 0.296, and 0.583 munits/5 μl of the extracts of bovine, human, and monkey livers, respectively.View full abstractDownload PDF (1136K) -
Sachiko IWAKI, Tohoru NAKAMURA, Jiro KOYAMA1986 Volume 99 Issue 5 Pages 1317-1326
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inhibitory effects of various 7-(aminoacyl)-4-methylcoumarinylamides (AA-MCA's). synthetic substrates for aminopeptidases, on phagocytosis of immune complexes by guinea pig peritoneal macrophages were investigated by measuring the intracellular uptake of sensitized 51Cr-sheep erythrocytes and 125I-α-amylase complexed with homologous IgG2 antibody. Among the AA-MCA's examined, MCA compounds of hydrophobic amino acids (Phe, Tyr, Leu, and Pro) were found to inhibit the intracellular uptake and digestion of immune complexes. Sucrose density gradient centrifugation of the homogenates of macrophages treated with the inhibitors for 1h at 37°C showed that they modulated the lysosomes, resulting in a decrease in buoyant density of the organella. These effects of the inhibitors on the buoyant density of the lysosomes as well as on the phagocytic activity of macro-phages disappeared upon removal of the inhibitors from the cells by washing. Since none of 7-amino-4-methylcoumarin, L-phenylalanine, and bestatin methyl ester could significantly inhibit the phagocytosis of immune complexes by macrophages, the MCA compounds of hydrophobic amino acids appear to inhibit the phagocytosis as a consequence of their lysosomotropic nature.View full abstractDownload PDF (831K) -
Shingo FUJISAKI, Tokuzo NISHINO, Hirohiko KATSUKI1986 Volume 99 Issue 5 Pages 1327-1337
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsopentenyl pyrophosphate (IPP) isomerase, farnesyl pyrophosphate (FPP) synthetase, octaprenyl pyrophosphate (OPP) synthetase and undecaprenyl pyrophosphate (UPP) synthetase were partially purified from Escherichia coli by DEAE-Toyopearl chromatography. FPP synthetase catalyzed the condensation of IPP with dimethylallyl pyrophosphate (DPP) as well as with geranyl pyrophosphate (GPP) to yield FPP as final product. OPP synthetase and UPP synthetase catalyzed the condensation of IPP with FPP to yield OPP and cis, trans-polyprenyl pyrophosphates (the C45-, C50, and C55-compound), respectively. Neither DPP nor GPP acted as a priming substrate for either enzyme. These four enzymes required Mg2+ or Mn2+ for their activities. UPP synthetase required also Triton X-100 for its activity. The addition of Triton X-100 enhanced OPP synthetase, but it did not affect IPP isomerase and FPP synthetase. It seems possible that the combination of the four enzymes ensures the in vivo synthesis of long-chain isoprenoids in E. coli.View full abstractDownload PDF (837K) -
Hideaki OTSUKA, Osamu OTSURU, Takeshi KASAMA, Akihiko KAWAGUCHI, Kazuo ...1986 Volume 99 Issue 5 Pages 1339-1344
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe stereochemistry of the alcohol moieties of 2, 3-alkanediol diacyl esters obtained from the Harderian gland of the Mongolian gerbil was investigated. There were five major 2, 3-alkanediols, C14-C22 (even carbon numbers), all having the erythro configuration as determined by GC-MS analysis of their isopropylidene derivatives in comparison with synthetic erythro- and threo-2, 3-hexadecanediols. 13C-NMR spectroscopy of the synthetic materials showed distinct differences of chemical shift at the C-1, C-3, and C-4 carbons, from which the native 2, 3-alkanediols were definitely determined to be in the erythro series. The absolute configurations of the C-2 and C-3 asymmetric centers were assigned as 2S and 3R, respectively, based on known 2S, 3R-octanediol.View full abstractDownload PDF (402K) -
Shuichi HORIE, Mitsui ISOBE, Tetsuya SUGA1986 Volume 99 Issue 5 Pages 1345-1352
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChanges in peroxisomal CoA pools in the liver of fasted, diabetic, high-fat diet-fed and clofibrate-treated rats were studied. Total-CoA increased slightly in the fasted group and markedly in the diabetic, high-fat and clofibrate-treated groups. Fractionation studies showed that changes in free CoA levels were much greater in peroxisomes than in mitochondria. The concentrations of CoAs were calculated from the contents of CoAs in organelles and the changes in volume of organelles under these conditions; the concentration of total CoA in peroxisomes was higher than that in cytosol, but lower than that in mitochondria. These changes were accompanied by an increase in the activity of peroxisomal β-oxidation.
The results obtained from these experiments indicate that the peroxisomal β-oxidation system is controlled not only at the enzyme level but also at the substrate or co-factor level.View full abstractDownload PDF (577K) -
Fuyuki IWASA, Koichi ISHIGURO1986 Volume 99 Issue 5 Pages 1353-1358
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA calmodulin-binding protein from sea urchin eggs consisting of two subunits (55 and 17K-daltons) was identified as a Ca2+-dependent phosphoprotein phosphatase similar to calcineurin in mammalian brain and to phosphatase 2B in skeletal muscle. Peptide mappings showed that the 55K subunit was different from 61K subunit of calcineurin, whereas the 17K subunit was similar to 19K subunit of calcineurin but different from calmodulin. The 55K+17K protein of sea urchin eggs dephosphorylated 32P-inhibitor-1 in a Ca2+- and calmodulin-dependent manner. Vmax and Km for inhibitor-1 in the presence of Ca2+ and calmodulin were 2, 100 pmol P1/min/mg and 2.7μm. Ca2+-dependent phosphatase activity for inhibitor-1 was detected in homogenates of both unfertilized and fertilized eggs, but was not detected in isolated cortices and mitotic apparatus.View full abstractDownload PDF (2502K) -
Seiji NAKAMURA, Yoshiaki NONOMURA1986 Volume 99 Issue 5 Pages 1359-1369
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCross-linked complex of gizzard myosin light chain kinase (MLCK) and calmodulin (CM) was produced by glutaraldehyde treatment of a mixture of these proteins in a high Ca2+ (0.1mM) solution. Although the specific activity was reduced, this complex showed MLCK activity in a Ca2+-independent manner, different from the original MLCK whose activity was Ca2+-dependent. Chlorpromazine, one of the CM antagonists, was no longer able to inhibit the MLCK activity of this complex. These observations support the previously proposed hypothesis on the regulatory mechanism of MLCK activity via Ca2+. This complex could be regarded as another kind of Ca2+-independent MLCK different from that obtained by chymotryptic digestion of MLCK (Walsh, M. P., Dabrowska, R., Hinkins, S., & Hartshorne, D. J. (1982) Biochemistry 21, 1919-1925).
This complex caused superprecipitation of gizzard actomyosin and enhanced actin-activated ATPase of myosin Ca2+-independently.View full abstractDownload PDF (2573K) -
Atsuhiko OOHIRA, Fumiko MATSUI, Hiroshi NOGAMI1986 Volume 99 Issue 5 Pages 1371-1376
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to investigate the involvement of cartilage proteoglycans in the pathogenesis of human congenital skeletal disorders, proteoglycans were extracted with 4M guanidine HCl from the iliac crest cartilage of children with various skeletal diseases; lysosomal storage diseases (group I), osteochondrodysplasias (group II) and controls (group III). The cartilage-type proteoglycan (PG-H) was purified and its chondroitin sulfate moiety was analyzed by digestion with chondroitinase-ABC. In group II and group III, the relative amounts of the unsaturated disaccharide products changed in an age-related manner; decrease (from 50% to 30%) of ΔDi-4S with a compensatory increase (from 40% to 60%) of ΔDi-6S with increasing age from 0 to 15 years. On the other hand, some cases in group I showed aberrant composition of the disaccharide products; a lower content of ΔDi-4S with a correspondingly higher content of ΔDi-6S. Patients in group I have clinically similar skeletal disorders, and the extent of the compositional abnormality seems to reflect the severity of the skeletal disorder. Therefore, one may consider that the aberrant composition of the glycosaminoglycans in PG-H is involved in the pathogenesis of the skeletal disorder of lysosomal storage diseases.View full abstractDownload PDF (422K) -
Motohiro MATSUURA, Yasuhiko KOJIMA, J. Yuzuru HOMMA, Yoshio KUMAZAWA, ...1986 Volume 99 Issue 5 Pages 1377-1384
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmong chemically synthesized analogues corresponding to the nonreducing sugar part of lipid A, we have found an analogue (GLA-27) which exhibits Limulus, mitogenic, polyclonal B cell activation (PBA), interferon-inducing, and tumor necrosis factor (TNF)-inducing activities but not pyrogenic activity. The structure of GLA-27 comprises 4-O-phosphono-D-glucosamine with tetradecanoyl and 3-tetradecanoyloxytetradecanoyl (C14-O-(C14)) groups as the 3-O- and 2-N-acyl substituents, respectively.
Derivatives of GLA-27 with different backbone structures, such as the 1-deoxy, 3-epimeric, 3-amino, and 1-deoxy-3-epimeric derivatives of glucosamine, were chemically synthesized, and their mediator-inducing activities such as interferon- and TNF-inducing activities were investigated in comparison with their B cell activation activities including mitogenic and PBA activities. Among these derivatives, a derivative with a 1-deoxyglucosamine backbone (GLA-40) exhibited stronger B cell activation activities than those of GLA-27 while the mediator-inducing activities of GLA-40 were weaker than those of GLA-27. In addition to these derivatives, stereoisomers of GLA-27 which possess the (R) and (S) forms of C14-O-(C14) as the 2-N-acyl substituent were also synthesized and their biological activities compared. The (S) isomer exhibited much stronger mediator-inducing activities than the (R) isomer. On the other hand, B cell activation activities of the (R) isomer were strong and those of the (S) isomer weak.
These results clearly demonstrate that mediator-inducing activities and B cell activation activities can be selectively expressed by modifying the structures of lipid A analogues.View full abstractDownload PDF (552K) -
Kiyoshi NOSE1986 Volume 99 Issue 5 Pages 1385-1391
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNuclear matrix was isolated from cultured human fibroblasts by extraction of nuclei with 2M NaCl. Electron microscopic observation on the isolated nuclear matrix revealed a fine network structure. The matrix fraction contained approximately 15% of total nuclear DNA and the matrix DNA was about 3- to 4-fold enriched in transcriptionally active collagen I (α2) gene sequences, whereas transcriptionally inactive β-globin gene sequences were not enriched.
The nuclear matrix contained two major proteins of 65, 000 and 45, 000 daltons (p1 5.9 and 5.6, respectively). The DNA-binding activity of these nuclear matrix proteins was examined by Western blotting or by nitrocellulose filter-binding assay using cloned specific gene probes. The results suggest that there is no base sequence specificity in the binding, and that protein species of 60, 000 to 200, 000 daltons showed DNA-binding activity. These results indicate that association of transcribing genes with the nuclear matrix may reflect the functional state of the genes and may not be determined solely by the base sequence specificity of DNA binding. The nuclear matrix protein of 65, 000 daltons was phosphorylated in vivo, and was the main substrate for protein kinase(s) associated with the nuclear matrix.View full abstractDownload PDF (2175K) -
Toshihide KOBAYASHI, Akinori OHTA, Isao SHIBUYA1986 Volume 99 Issue 5 Pages 1393-1400
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEscherichia coli mutants harboring the pss-1 allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant. For this purpose, a pair of E. coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpK1, and phoA8). Pulse- and uniform-labeling of phospholipids with 32P at 42°C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperaturesensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant. The phospholipid synthesis and contents in the pssr+ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose. The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E. coif membranes. Possible determinants for the phospholipid composition of the wild-type E. coli cells are also discussed on the basis of the present observations.View full abstractDownload PDF (666K) -
Toshiko MATSUBARA, Akira HAYASHI1986 Volume 99 Issue 5 Pages 1401-1408
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA series of glycosphingolipids and phosphonoglycosphingolipid containing only galactose as the sugar component were isolated from the marine snail, Chlorostoma argyrostoma turbinatum. The structures of these lipids were studied by methylation analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, and characterized as follows: the glycosphingolipids (a) galactosyl β(1-1)ceramide, (b) galactosyl β(1-6)galactosyl β(1-1)ceramide, (c) galactosyl β(1-6)galactosyl β(1-6)galactosyl β(1-1)ceramide and (d) galactosyl β(1-6)galactosyl β(1-6)galactosyl β(1-6)galactosyl β(1-1)ceramide, and phosphonoglycosphingolipid N-methylaminoethylphosphonyl galactosyl(1-1)ceramide.
The main molecular species of the ceramide moiety were hexadecanoyl-octadecasphingenine and hydroxyhexadecanoyl-octadecasphingadienine in all of these sphingolipids.View full abstractDownload PDF (523K) -
Hiroshi KAWASAKI, Yasuyuki KUROSU, Hisataka KASAI, Toshiaki ISOBE, Tsu ...1986 Volume 99 Issue 5 Pages 1409-1416
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe limited proteolysis of bovine brain calmodulin with trypsin in the presence or absence of various metal ions was reinvestigated in detail by HPLC. With metal ion-free calmodulin, limited proteolysis occurred at Arg 37 and Arg 106 with a cleavage ratio of 1 to 5, resulting in fragments consisting of residues 1-37, 38-148, 1-106 and 107-148. Fragments 1-37 and 107-148 accumulated under metal ionfree conditions. In the presence of calcium ions, the susceptibility of these sites to trypsin decreased and limited proteolysis occurred at Lys 77 as already reported by other workers. Fragment 78-148 accumulated, whereas fragment 1-77 was unstable under calcium-bound conditions, giving smaller peptides. Upon binding of manganese ions, calmodulin underwent a change of susceptibility to trypsin, resulting in cleavage at Lys 77, as observed for calcium-bound calmodulin. In the presence of zinc or magnesium ions, calmodulin was cleaved at the same sites as metal ion-free calmodulin under conditions where calmodulin would be expected to bind the respective ions.View full abstractDownload PDF (566K) -
Hidenari TAKAHARA, Hiroomi OKAMOTO, Kiyoshi SUGAWARA1986 Volume 99 Issue 5 Pages 1417-1424
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1, 800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported.
The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3×10-7 M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity. These results suggest that peptidylarginine deiminase interacts with immobilized STI in almost the same manner as with the soluble substrate, and the Ca2+ requirement can be explained in terms of activation of the substrate-binding site of the enzyme.View full abstractDownload PDF (1018K) -
Taiko AKAO, Teruaki AKAO, Masao HATTORI, Tsuneo NAMBA, Kyoichi KOBASHI1986 Volume 99 Issue 5 Pages 1425-1431
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRuminococcus sp. PO1-3 obtained from human intestinal flora is able to reduce dehydrocholate as well as 3-ketoglycyrrhetinate. From this bacterium dehydro-cholate- and 3-ketoglycyrrhetinate-reducing activities were purified one thousandfold together with 3-ketocholanate-reducing and 3β-hydroxyglycyrrhetinate (glycyrrhetic acid) oxidizing activities by means of M_??_trex Red A, Sephadex G-200 and Octyl-Sepharose column chromatography. The purified enzyme catalyzed the reduction of dehydrocholic acid to 3β-hydroxy-7, 12-diketocholanic acid and of 3-ketocholanic acid to 3β-hydroxycholanic acid. Studies on substrate specificity revealed that the enzyme had absolute specificity for the β-configuration of a hydroxyl group at the 3 position of bile acid and steroids having no double bond in the A/B ring. This enzyme was neither β-hydroxysteroid dehydrogenase [EC 1.1.1.51] nor 3β-hydroxy-Δ5-steroid dehydrogenase [EC 1.1.1.145], but a novel type of enzyme, defined as 3β-hydroxysteroid dehydrogenase.View full abstractDownload PDF (555K) -
Kazuhiro KOHAMA, John KENDRICK-JONES1986 Volume 99 Issue 5 Pages 1433-1446
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (>95%), in a high yield (about 10mg/100g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500, 000 Mr myosin).
Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when the Ca2+ concentration was increased to 50μM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3μM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+.
The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2μM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin.
The involvement of this myosin-linked regulatory system in the Ca2+-control of cytoplasmic streaming is discussed.View full abstractDownload PDF (2488K) -
Ikuo NISHIDA, Akihiko KAWAGUCHI, Mitsuhiro YAMADA1986 Volume 99 Issue 5 Pages 1447-1454
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThiolactomycin, an antibiotic with the structure of (4S)-(2E, 5E)-2, 4, 6-trimethyl-3-hydroxy-2, 5, 7-octatriene-4-thiolide, selectively inhibits type II fatty acid synthases. The mode of the thiolactomycin action on the fatty acid synthase system of Escherichia coli was investigated. Of the six individual enzymes of the fatty acid synthase system, [acyl-carrier-protein] (ACP) acetyltransferase and 3-oxoacyl-ACP synthase were inhibited by thiolactomycin. On the other hand, the other enzymes were not affected by this antibiotic. The thiolactomycin inhibition of the fatty acid synthase system was reversible. As to ACP acetyltransferase, the inhibition was competitive with respect to ACP and uncompetitive with respect to acetyl-CoA. As to 3-oxoacyl-ACP synthase, the inhibition was competitive with respect to malonyl-ACP and noncompetitive with respect to acetyl-ACP. The thiolactomycin action on the fatty acid synthase system was compared with that of cerulenin.View full abstractDownload PDF (589K) -
Satoshi SHIOJIRI, Rikiro FUKUNAGA, Yoshitake ICHII, Yoshihiro SOKAWA1986 Volume 99 Issue 5 Pages 1455-1464
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS17S poly(A)+RNA, which hybridized to an oligonucleotide complementary to a part of the partial cDNA (E1cDNA) (Merlin et al. (1983) Proc. Natl. Acad. Sci. U.S. 80, 4904) for 2-5A synthetase, was isolated from interferon-treated human KB cells and used for cDNA cloning. Several overlapping cDNAs were cloned by using the oligonucleotide as a probe. Two of them were joined at their overlapping region, resulting in a cDNA (22-1 cDNA) of 1.4 kb containing a long open reading frame. When the cDNA was expressed in COS-7 cells with an eukaryotic promoter, active 2-5A synthetase was produced and localized mainly in the cytoplasm. The 5'-proximal ATG in 22-1 cDNA is followed immediately by another ATG. This second ATG was assumed to work as the initiator codon. If so, this enzyme comprises 363 amino acids.View full abstractDownload PDF (1607K) -
Takao KODAMA, Kazuhiro FUKUI, Kaoru KOMETANI1986 Volume 99 Issue 5 Pages 1465-1472
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Malachite Green method for determination of inorganic phosphate (P1) (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361-366) was modified to measure P1 in the range of 0.2-15nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2g of sodium molybdate, 0.3g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in I liter of 0.7M HCl, and the absorbance at 650nm is then measured after a 35-40 min incubation at 25°C.
Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately using relatively low concentrations of native myosin and its subfragment-1. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8-0.9mol per site in 0.1M KCl, 10mM MgCl2, pH 8.0 at 25°C for fresh enzyme preparations.View full abstractDownload PDF (601K) -
Ronald H. LOCKER, David J. C. WILD1986 Volume 99 Issue 5 Pages 1473-1484
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA wide range of phyla have been surveyed by SDS-PAGE for the new large proteins of the myofibril. Connectin (or titin) appears to be widely distributed. It is seen as a band of constant intensity and mobility in vertebrate striated muscle, but is absent from smooth muscle. It appears in more variable amounts, in a form of constant but greater mobility in many invertebrates: worms, molluscs (adductor but not gastropod feet), insects, a myriapod, and even in human blood platelets. Nebulin shares the same distribution in vertebrate muscles except for its notable absence in all heart muscle examined. It too is found in many invertebrates, not always with titin. It has been found in a worm, molluscs (adductor and gastropod feet), insects, crustaceans and an echinoderm. The mobility of nebulin varies within the vertebrates and more so between invertebrates (where, as with titin, it is greater). The isoforms of filamin in skeletal, cardiac, and smooth muscles of vertebrates are recorded. C-protein in rabbit muscles has four isoforms: white, α-red (X-protein), β-red, and cardiac.View full abstractDownload PDF (1978K) -
Di Hua HU, Sumiko KIMURA, Koscak MARUYAMA1986 Volume 99 Issue 5 Pages 1485-1492
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing an SDS gel electrophoresis method, connectin, very high molecular weight (_??_106 dalton) protein, was detected in an SDS extract of whole tissues of various types of muscles of vertebrates and invertebrates. Connectin bands were clearly recognized in all the types of striated muscles (skeletal and cardiac) of the vertebrates examined: rabbit, chicken, turtle, snake, newt, frog, and fish. This was also the case with skeletal muscle of prochordate, Amphioxus. In invertebrates, the situation was much complicated. Connectin-like protein bands were detected in C. elegans (nematode), but not in earthworm (annelid). Smaller sizes of proteins (_??_106) were faintly found in molluscan adductor muscles. In arthropods, connectinlike proteins were clearly detected in some muscles (e.g., claw muscles of crab and crayfish; leg muscles of several insects) but not at all in other muscles (e.g., tail muscles of crayfish and shrimp; thoracic muscles of some insects). These peculiar observations might be related to the presence of such specific elastic proteins as projectin in honeybee flight muscle. The present study has revealed that connectin is an elastic protein of vertebrate striated muscle, skeletal and cardiac muscles.View full abstractDownload PDF (1636K) -
Ryota KUROKI, Hidenori YAMADA, Taiji IMOTO1986 Volume 99 Issue 5 Pages 1493-1499
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n=2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.View full abstractDownload PDF (592K) -
Yuji GOTO, Kozo HAMAGUCHI1986 Volume 99 Issue 5 Pages 1501-1511
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformation of the constant fragment of the immunoglobulin light chain in which the intrachain disulfide bond is replaced by the bond S-Hg-S (CL, Hg fragment). was as compact as that of the intact CL fragment, but its stability to guanidine hydrochloride was lower than that of the intact CL fragment [Goto, Y. & Hamaguchi, K. (1986) Biochemistry in press]. The kinetics of reversible unfolding and refolding of the CL-Hg fragment by guanidine hydrochloride were studied and compared with those for the intact CL and reduced CL fragments [Goto, Y. & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910, 911-926]. The unfolding kinetics were explained on the basis of a three-species mechanism, U1_??_U2_??_F, where U1 and U2 are respectively slow-folding and fast-folding species of unfolded protein, and F is folded protein. However, an additional isomerization, though its contribution to the overall reaction process is small, had to be taken into account to explain the refolding kinetics. The kinetic properties of interconversion between U1 and U2 were similar to those for the intact CL and reduced CL fragments. This suggested that the same prolyl residue is involved in the isomerization reactions in the unfolded states of the intact CL, reduced CL, and CL-Hg fragments. The rate constant for the unfolding process, F to U2, was about 20 times greater than those for the intact CL and reduced CL fragments, while the rate constant for the refolding process, U2 to F, lay between the values for the intact CL and the reduced CL fragment. The free energy profiles of unfolding and refolding of the intact CL, reduced CL, and CL-Hg fragments were compared.View full abstractDownload PDF (895K) -
Shoji SHIMOMURA, Toshihiro SOTOBAYASHI, Masamitsu FUTAI, Toshio FUKUI1986 Volume 99 Issue 5 Pages 1513-1524
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21, 000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20, 000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the β-structures, and shows no α-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.View full abstractDownload PDF (2191K) -
The Small Subunits of Calcium Dependent Proteases with Different Calcium Sensitivities are IdenticalHiroshi KAWASAKI, Shinobu IMAJOH, Seiichi KAWASHIMA, Hiroaki HAYASHI, ...1986 Volume 99 Issue 5 Pages 1525-1532
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe small subunits of two calcium dependent proteases from rabbit with different calcium sensitivities were isolated by high performance liquid chromatography (HPLC) and their properties were compared. The isolated subunits were indistinguishable on polyacrylamide gel electrophoresis and HPLC. Their amino acid compositions were identical, and the peptides obtained on their digestion with lysylendopeptidase showed identical peptide maps on HPLC. Furthermore, the amino acid compositions and partial amino acid sequences of the corresponding peptides purified by HPLC were the same. These results indicate that the two calcium protease isozymes possess the same small subunit.View full abstractDownload PDF (549K) -
Jun UTSUMI, Shojiro YAMAZAKI, Kazuo HOSOI, Hirohiko SHIMIZU, Ken KAWAG ...1986 Volume 99 Issue 5 Pages 1533-1535
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformations of fibroblast and E. coli-derived recombinant human interferon-βs were studied by circular dichroism and nuclear magnetic resonance spectroscopy in the acidic pH region of 4.6 to 1.6. Both interferons have very similar conformations with high α-helix contents (_??_70%). These results suggest that glycosylation does not appreciably change the conformation of human interferon-β. Moreover, a slow conformational change is observed below pH 2.0, which induces the disruption of β-sheets.View full abstractDownload PDF (247K) -
Daisuke TSURU, Shinya SHIMADA, Shinsaku MARUTA, Tadashi YOSHIMOTO, Koh ...1986 Volume 99 Issue 5 Pages 1537-1539
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSScytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to G1u53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.View full abstractDownload PDF (244K) -
Nobuyuki NUKINA, Yasuo IHARA1986 Volume 99 Issue 5 Pages 1541-1544
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPaired helical filaments (PHF) are unusual neuronal fibers which accumulate progressively in the brain in Alzheimer's disease (AD). The insolubility of PHF in various kinds of solvents enabled us to obtain highly purified PHF, but prevented the application of conventional analytical methods to identify their components. Here we report that antibodies against purified PHF recognize tau protein, a brainspecific microtubule-associated protein, suggesting that a portion of PHF is tau protein.View full abstractDownload PDF (1471K) -
Kanenobu KUBO, Toshio TAKAGI1986 Volume 99 Issue 5 Pages 1545-1548
Published: May 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe separation of spinach thylakoid chlorophyll-proteins on polyacrylamide gel electrophoresis at 0°C in the presence of dodecyl sulfate is markedly influenced by the kind of surfactant cation in the media used for solubilization and electrophoresis. The mode of separation can thus be modulated through the cation selection. Three kinds of alkanolammonium dodecyl sulfates were tested and their abilities as to the dissociation of the molecular assemblies of chlorophyll-proteins were found to decrease in the following order: tris(hydroxymethyl)methylammonium, triethanolammonium and triisopropanolammonium. Comparison of the electrophoretic patterns obtained with different kinds of cation may help clarify the hierarchy of the molecular assemblies of chlorophyll-proteins.View full abstractDownload PDF (1023K) -
1986 Volume 99 Issue 5 Pages 1549a
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (19K) -
1986 Volume 99 Issue 5 Pages 1549b
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (19K)
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