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Volume 112 (1992)
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Volume 112, Issue 6
Displaying 1-25 of 25 articles from this issue
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Kwang-Hoon Kong, Hideshi Inoue, Kenji Takahashi1992 Volume 112 Issue 6 Pages 725-728
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe four residues of human glutathione S-transferase Pl-1 whose counterparts were indicated by X-ray crystallography to reside in the GSH-binding site of pig glutathione S-transferase P1-1 were individually replaced with threonine or alanine by site-directed mutagenesis to obtain mutants R13T, K44T, Q51A, and Q64A. The kinetic parameters, susceptibilities to an inhibitor, S-hexyl-GSH, and affinities for GSH-Sepharose of the latter were compared with those of the wild-type enzyme, and pKa of the thiol group of GSH bound in R13T was shown to be equivalent to that in the wild type. From the results, Lys44, Gln51, and GLn64 were deduced to contribute to the binding of GSH. On the other hand, Arg13 seems to be essential for the enzymatic activity as mainly involved in the construction of a proper structure of the active site.View full abstractDownload PDF (481K) -
Takahiro Miyauchi, Masahiro Ariki, Hirofumi Usui, Kentaro Semba, Yumik ...1992 Volume 112 Issue 6 Pages 729-732
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour tyrosine-protein kinases that reacted with antibodies specific to p62c-yes, p60c-src, p60c-src+, and p59fyn, respectively, were solubilized from a rat brain particulate fraction and separated by casein-Toyopearl column chromatography. Possible p59fyn, with a pI of 6.5, was purified 490-fold as a single 59-kDa protein band on SDS-PAGE. The purified enzyme contained almost no phosphotyrosine residues but was autophosphorylated with Mg2+, ATP exclusively at tyrosine residues, with a concomitant increase in the kinase activity toward tyrosine-glutamate (1:4) copolymers. The rate of the copolymer phosphorylation was proportional to the square of the enzyme concentration, suggesting activation through intermolecular catalysis. In the presence of Mn2+, however, the reaction showed a firstorder dependence on the enzyme concentration.View full abstractDownload PDF (2822K) -
Sakae Shimizu, Tsuneo Imanaka, Tatsuya Takano, Shoji Ohkuma1992 Volume 112 Issue 6 Pages 733-736
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that novel Mg2+-ATPases were induced in rat liver peroxisomes by clofibrate administration and that these activities consisted of at least two types of enzymes, N-ethylmaleimide (NEM)-sensitive and -resistant. Here we present evidence that neither of these major peroxisomal ATPases is associated with the 70-kDa peroxisomal membrane protein (PMP70), because: (i) proteinase K treatment of peroxisomes resulted in inactivation of only NEM-sensitive ATPase, whereas disappeared PMP70 completely; (ii) NEM-sensitive ATPase activity was barely immunoprecipitated with anti-PMP70 IgG; (iii) the solubilized ATPases behaved differently from PMP70 on native PAGE; and finally (iv), the major peroxisomal ATPases were separated from PMP70 on gel filtration chromatography.View full abstractDownload PDF (2797K) -
Masanori Mitta, Hiroshi Ohnogi, Akira Yamamoto, Ikunoshin Kato, Fumio ...1992 Volume 112 Issue 6 Pages 737-742
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA encoding the complete amino acid sequence of aminoacylase 1 (N-acylamino acid aminohydrolase, ACY-1) [EC 3. 5. 1. 14], a dimeric metalloprotein having two Zn2+ in the molecule, which catalyzes the deacylation of N-acylated L-amino acids except L-aspartic acid, has been isolated from porcine kidney λgt10 cDNA library and sequenced. From sequence analysis of the cDNA and the N- and C-terminal amino acid analyses of the purified protein, it is deduced that porcine kidney ACY-1 consists of two identical subunits (Mr 45, 260), each of which consists of a single chain of 406 amino acids with acetylalanine at the N-terminus. A cDNA encoding porcine liver ACY-1 was also cloned. The amino acid sequence deduced from the nucleotide sequence of the cDNA from porcine liver was identical to that deduced for porcine kidney ACY-1. Northern blot analysis suggested that ACY-1 is more highly expressed in kidney than in liver. Comparison of the amino acid sequence of porcine ACY-1 with those of other Zn2+-binding metalloenzymes showed no significant homologies in either the overall sequence or the consensus sequences for the metal binding sites. This indicates that ACY-1 is a new type of metalloprotein.View full abstractDownload PDF (668K) -
Shin-ichi Ohnuma, Tanetoshi Koyama, Kyozo Ogura1992 Volume 112 Issue 6 Pages 743-749
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFactors that affect the termination of isoprenoid chain elongation catalyzed by prenyltransferase were investigated. The chain-length distribution of reaction products of solanesyl diphosphate synthase [EC 2. 5. 1. 11] homogeneously purified from Micrococcus luteus changed dramatically according to the concentration of the complex formed between isopentenyl diphosphate and Mg2+ (IPP-Mg) in the reaction mixture. However, the concentration of the complex between farnesyl diphosphate and Mg2+ (FPP-Mg), the priming substrate for this synthase, did not affect the product distribution, provided that the concentration of IPP-Mg was maintained at a certain level. Thus, the level of IPP-Mg is decisive in affecting the chain length distribution of the products of the prenyltransferase reaction, and the Mg2+-dependent variability of product specificity so far observed can now be understood in terms of the effect of IPP-Mg concentration.View full abstractDownload PDF (694K) -
Hiroshi Nakamura, Yasuaki Nakasaki, Naruto Matsuda, Munekazu Shigekawa1992 Volume 112 Issue 6 Pages 750-755
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe characterized the interaction of 2, 5-di (tert-butyl)-1, 4-benzohydroquinone (tBuBHQ) with the sarcoplasmic reticulum (SR) Ca2+-ATPase from rabbit fast-twitch skeletal and canine cardiac muscles by examining the effect of this agent on the ATPase reaction. tBuBHQ at less than 10μM inhibited ATP hydrolysis by both isoforms of Ca2+-ATPase by up to 80 and 90%, respectively. The half maximal inhibition of these enzymes was observed at about 1.5μM tBuBHQ. Thus, this agent potently inhibits the fast-twitch skeletal and slow-twitch skeletal/cardiac isoforms of SR Ca2+-ATPase. tBuBHQ at 5-10μM inhibited the rate of decomposition of the phosphoenzyme intermediate (EP), measured as a ratio between ATPase activity and the EP level in the steady state, by 35-40%. It also inhibited formation of EP by decreasing the rate of Ca2+ binding to the Ca2+-deficient, nonphosphorylated enzyme to about 1/8 of the control value. These results indicate that tBuBHQ has at least two sites of action in the reaction sequence for the SR Ca2+-ATPase.View full abstractDownload PDF (632K) -
Takashi Sato, Tsutomu Hashizume, Tatsuzo Fujii1992 Volume 112 Issue 6 Pages 756-761
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe role of phosphatidic acid (PA) in the signal transduction system of platelets was studied using 1-stearoyl 2-arachidonoyl PA (PASA). When PASA was added to rabbit platelets, aggregation occurred. BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase, as well as p-bromophenacyl bromide and mepacrine, inhibitors of phospholipase A2, inhibited the aggregation induced by low concentrations of PASA, but not that induced by high concentrations. PASA also stimulated, in a dose-dependent manner, arachidonic acid liberation, lysophosphatidylcholine and diacylglycerol formation, and mobilization of intracellular Ca2+; all of which were dependent on the presence of Ca2+ in the outer medium. The arachidonic acid liberation was inhibited by p-bromophenacyl bromide or mepacrine, while diacylglycerol formation by low concentrations of PASA was inhibited by BW755C. With platelet membrane fractions or with the platelets made permeable to Ca2+ by pretreatment with ionomycin, PASA caused arachidonic acid liberation in the presence of Ca2+. Furthermore, PASA enhanced the activity of phospholipase A2 partially purified from platelet cytosol acting on 1-palmitoyl-2-[14C]arachidonoyl-glycerophosphoethanolamine. These results provide evidence that PASA preferentially potentiates the activation of phospholipase A2 in cooperation with Ca2+, suggesting that PA acts as a positive feedback regulator to potentiate the activation of phospholipase A2 and contributes to the amplification of platelet activation.View full abstractDownload PDF (765K) -
Noriko Takeuchi, Toru Nakamura, Fumito Takeuchi, Eikichi Hashimoto, Hi ...1992 Volume 112 Issue 6 Pages 762-767
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50, value was 4.4μg/ml (8.5μM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100μg/ml (>170μM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its K1 value was 6.3μM (K1 values of daunorubicin and doxorubicin were 0.89 and 0.15mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50, values were 0.49μg/ml (0.95μM), 1.8μg/ml (3.5μM), and 0.82μg/ml (1.6μM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10μg/ml. On the other hand, brief exposure (5min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50, value of 52ng/ml (0.1μM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10μg/ml mitoxantrone for 5min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.View full abstractDownload PDF (1788K) -
Maria Ruzzene, Fabio Vianello, Arianna Donella-Deana, Renzo Deana1992 Volume 112 Issue 6 Pages 768-774
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19μM). Enzyme activity was maximal in the presence of 10mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodiesraised against recombinant α, but not β, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12μM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30mM), and NaCl (2O-400mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.View full abstractDownload PDF (3367K) -
Ryuichi Tatsumi, Koui Takahashi1992 Volume 112 Issue 6 Pages 775-779
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen chicken breast muscle myofibrils were treated with a solution containing 0.1mM CaCl2 and 30μg of leupeptin/ml, nebulin filaments were fragmented into 200-, 180-, 40-, 33-, and 23-kDa subfragments. All the subfragments except the 180-kDa one were released from the myofibrils. The fragmentation of nebulin filaments seems to be induced by the binding of large amounts of calcium ions. Similar changes took place in nebulin filaments in post-mortem skeletal muscle. It has been proposed that nebulin co-exists with thin (actin) filaments and participates in stabilizing their organization [Wang, K. & Wright, J. (1988) J. Cell Biol. 107, 2199-2212]. Thus, the above result suggests that Ca-induced fragmentation of nebulin filaments destabilizes the organization of thin filaments and is a key factor in meat tenderization during post-rigor aging.View full abstractDownload PDF (1529K) -
Ryuichi Tatsumi, Akihito Hattori, Koui Takahashi1992 Volume 112 Issue 6 Pages 780-785
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNebulin, which forms a long inextensible filament in sarcomeres, was fragmented into 200-, 180-, 40-, 33-, and 23-kDa subfragments on treatment with 0.1mM CaCl2. The subfragments released from myofibrils were successfully purified by immunoaffinity column chromatography. The 200-, 40-, 33-, and 23-kDa subfragments were released from myofibrils and occupied 80% of the nebulin filaments. The remainder comprised the 180-kDa subfragment bound to the myofibrils. There is a possibility that an entire nebulin filament is constructed from the 200-, 180-, 40-, 33-, and 23-kDa subfragments. We have developed a new “fluorescence-method” to detect the binding of calcium ions to a protein using quin2, and clarified that nebulin is a calcium-binding protein, and that calcium ions bind to the 200-, 40-, and 23-kDa subfragments. Nebulin filaments are probably fragmented on the binding of large amounts of calcium ions to the 200-, 40-, and 23-kDa subfragments.View full abstractDownload PDF (4375K) -
Hisao Kobayashi, Akihiro Inoue, Takashi Mikawa, Hideto Kuwayama, Yoshi ...1992 Volume 112 Issue 6 Pages 786-791
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155, 000 and 130, 000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the aminoterminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.View full abstractDownload PDF (2996K) -
Shigetoshi Aono, Hiromichi Kurita, Seiichi Uno, Ichiro Okura1992 Volume 112 Issue 6 Pages 792-795
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13, 000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.View full abstractDownload PDF (1077K) -
Hiroshi Hayashi, Hiroshi Kondo1992 Volume 112 Issue 6 Pages 796-799
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCrystals of a complex of chicken gizzard G-actin and DNase I were soaked in a solution of radioactive 4-hydroxymercuribenzoate (MB). The soaked crystals, which contained 0.93 mol of MB per mol of G-actin, were dissolved in “G-buffer” and digested with trypsin, and the resulting peptides were fractionated by thin-layer chromatography. The MB is exchangeable between peptides that contain cysteine residues, but the data obtained here suggested that MB attached to the cysteine residue at the 373rd position of the G-actin molecule.View full abstractDownload PDF (3342K) -
Masaru Tanokura, Masamoto Imaizumi, Kazuhiro Yamada, Fumie Shiraishi, ...1992 Volume 112 Issue 6 Pages 800-803
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTroponin C was isolated from the skeletal muscle of bullfrog (Rana catesbeiana), and its relative molecular mass was estimated to be 18, 000 by SDS/polyacrylamide gel electrophoresis. In its amino acid composition, bullfrog troponin C was similar to that of the frog (Rana esculenta) but different from that of rabbit. Its ultraviolet spectrum was consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca2+-loaded form vs. the metal-free form indicated that the single Tyr residue and some Phe residues in the bullfrog troponin C molecule were affected by the conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14mM Tris and 90mM glycine, the metal-free and Mg2+-loaded forms migrated slower than the Ca2+-loaded form. The property is shared by rabbit troponin C but not parvalbumins or calmodulin. The ATPase activity of CDTA-treated myofibrils reconstituted with bullfrog troponin C showed the same Ca2+- and Sr2+-sensitivity as that of those reconstituted with rabbit troponin C. Bullfrog troponin C is, thus, physiologically the same as rabbit troponin C, in spite of several marked differences in their physicochemical properties.View full abstractDownload PDF (1342K) -
Kyosuke Sato, Yasuzo Nishina, Kiyoshi Shiga1992 Volume 112 Issue 6 Pages 804-810
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSApoprotein of electron-transferring flavoprotein (ETF) reacts with FAD as follows: A*_??_A, A+FAD_??_holoETF. Two different forms of apoETF (A* and A) convert into each other and only one of them, A, can associate with FAD [Sato, K. et al. (1991) J. Biochem. 109, 734-740]. In the present study, the reactions between apoETF and ATP, ADP, AMP, riboflavin, or FMN were investigated. It was revealed that all three adenine nucleotides bind with apoETF with the same kinetic reaction scheme as FAD, and compete with FAD. These results suggest that the nucleotides bind to A with the same location as the ADP part of FAD in holoETF and that the ADP-binding site of apoETF is generated upon conversion from A* to A. Neither riboflavin nor FMN bound to apoETF regardless of the presence or absence of the nucleotides, indicating that the ADP part of the FAD molecule is essential to the incorporation of the isoalloxazine ring into ETF. The binding rate constant of FAD to A was 1/20 of that of ADP while the dissociation rate constant was 1/1, 000. This indicates that the riboflavin part of FAD inhibits the binding of FAD by steric hindrance, while after the binding, it stabilizes the complex.View full abstractDownload PDF (809K) -
Toshio Tanaka, Satoshi Yamamoto, Makoto Taniguchi, Hideyuki Hayashi, S ...1992 Volume 112 Issue 6 Pages 811-815
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAspartate aminotransferase (AspAT) [EC 2. 6. 1. 1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.View full abstractDownload PDF (1748K) -
Masahiro Kai, Takahiro Yano, Hideyuki Tamegai, Yoshihiro Fukumori, Tat ...1992 Volume 112 Issue 6 Pages 816-821
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-β-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ionexchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595nm in the oxidized form and at 440 and 595nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked γ band appeared at 429 and 438nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53, 000, 22, 000, and 17, 000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169, 000 on the basis of the results obtained by gel filtration, while the molecular weight per heme a was estimated to be 83, 700. The copper content of the oxidase was 1.01g atom per mol of heme a. Therefore, the cytochrome seemed to contain one molecule of heme α and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5. The enzyme also oxidized the reduced form of rusticyanin, and the optimum pH of the reaction was 4.0. In the enzymatic oxidation of ferrocytochrome c-552 at pH 4.0, Km of the oxidase for the ferrocytochrome was determined to be 17μM, and Vmax of the enzyme to be 6.3 mol of ferrocytochrome c oxidized per mol of heme a of the oxidase per s. In the oxidation of the reduced form of rusticyanin at pH 4.0, Km of the oxidase for the protein was approximately 600μM and Vmax of the enzyme was approximately 50 mol of reduced rusticyanin oxidized per mol of heme α of the oxidase per s. Sulfate ions stimulated the enzymatic oxidation of these electron donors. It seems interesting that a-type cytochrome c oxidase can oxidize a copper protein at a high molecular activity.View full abstractDownload PDF (1816K) -
Michikazu Abe, Yumiko Shirakata, Dai Sato, Kazuhiko Konishi, Tsuyoshi ...1992 Volume 112 Issue 6 Pages 822-827
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (≤20°C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100mg protein from 100g of the scallop muscle. Ca2+-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca2+-ATPase were observed at a pH of about 7.0 and temperature of 20-30°C, and their values were about 2μmol Ca2+/mg of protein/min and about 3μmol ATP hydrolysis/mg of protein/min, respectively. At 0°C, 10-20% of these activities was maintained, while at 37°C, the activities were irreversibly lost. The Ca2+-ATPase activity was half-maximally activated at about 0.3μM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of _??_10μM and 0.1-0.3mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80°C with retention of function for about a year.View full abstractDownload PDF (2111K) -
Kinuko Kimura, Shigeyoshi Sugano1992 Volume 112 Issue 6 Pages 828-833
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInstability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP). The crude extract from B. subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract. To understand the structural basis of the functional change, oxidative modification of B. subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts. The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme. The loss of catalytic activity was proportional to the weakening of subunit interactions. B. subtilis glutamine synthetase was protected from oxidative modification by either 5mM Mn2+ or 5mM Mn2+ plus 5mM ATP, but not by Mg2+. The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein. These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme. Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein. In B. subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.View full abstractDownload PDF (3093K) -
Michio Shinoda, Akira Hara, Toshihiro Nakayama, Yoshihiro Deyashiki, H ...1992 Volume 112 Issue 6 Pages 834-839
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDihydrodiol dehydrogenase from pig liver was inactivated by diethylpyrocarbonate (DEP) and by rose bengal-sensitized photooxidation. The DEP inactivation was reversed by hydroxylamine and the absorption spectrum of the inactivated enzyme indicated that both histidine and tyrosine residues were carbethoxylated. The rates of inactivation by DEP and by photooxidation were dependent on pH, showing the involvement of a group with a pKa of 6.4. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of one histidine residue per active site. The inactivation by the two modifications was partially prevented by either NADP(H) or the combination of NADP+ and substrate, and completely prevented in the presence of both NADP+ and a competitive inhibitor which binds to the enzyme-NADP+ binary complex. The DEP-modified enzyme caused the same blue shift and enhancement of NADPH fluorescence as did the native enzyme, suggesting that the modified histidine is not in the coenzyme-binding site of the enzyme. The results suggest the presence of essential histidine residues in the catalytic region of the active site of pig liver dihydrodiol dehydrogenase.View full abstractDownload PDF (714K) -
Michio Shinoda, Akira Hara, Toshihiro Nakayama, Yoshihiro Deyashiki, S ...1992 Volume 112 Issue 6 Pages 840-844
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDimeric dihydrodiol dehydrogenases from pig liver, monkey kidney, and rabbit lens were inhibited more potently by 4-hydroxyphenylketones such as 4-hydroxybenzaldehyde, 4-hydroxyphenylglyoxal, and 4-hydroxyacetophenone than by isoascorbate and ascorbate, known inhibitors of the enzymes. No significant inhibition was observed with 2- or 3-hydroxyphenylketones, phenylketones with a functional group other than a hydroxy group at the 4-position, and 4-hydroxyphenyl derivatives without a carbonyl group. The steady-state kinetic analyses of the inhibition of the pig liver enzyme indicated that the 4-hydroxyphenylketones, similarly to ascorbate and its epimer, bound to an enzyme-NADP+ binary complex as competitive inhibitors with respect to dihydrodiol substrate. The inhibition by the 4-hydroxyphenylketones was uncompetitive with respect to isoascorbate, and the addition of one of the 4-hydroxyphenylketones or isoascorbate with NADP+ afforded a great protective effect against inactivation of the enzyme by diethylpyrocarbonate or by heat treatment, which indicates that 4-hydroxyphenylketones and isoascorbate bind at the same site in or near the active center of the enzyme. The structural comparison of 4-hydroxybenzaldehyde and ascorbate suggests that the hydroxy group at C-5, carbonyl group at C-1 and lactone ring of ascorbate are important for the binding to the enzyme.View full abstractDownload PDF (599K) -
Tatsuo Kurihara, Mitsuyoshi Ueda, Naomi Kamasawa, Masako Osumi, Atsuo ...1992 Volume 112 Issue 6 Pages 845-848
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence of two types of thiolases, acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase, was demonstrated in peroxisomes of n-alkane-grown Candida tropicalis [Kurihara, T., Ueda, M., & Tanaka, A. (1989) J. Biochem. 106, 474-478], while acetoacetylCoA thiolase was also shown to be present in cytosol. The activity of the enzyme in cytosol was constant irrespective of culture conditions, while the peroxisomal enzyme was inducibly synthesized in the alkane-grown yeast cells. These results indicate that peroxisomal acetoacetyl-CoA thiolase participates in alkane degradation, while the cytosolic enzyme is associated with other fundamental metabolic processes, probably sterol biosynthesis, because this enzyme can catalyze the first step of the sterol biosynthesis. 3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, a key regulatory enzyme of sterol biosynthesis, was found to be localized exclusively in microsomes of the alkane-grown yeast cells. These results suggest that yeast peroxisomes do not contribute to sterol biosynthesis, unlike the case of mammalian cells.View full abstractDownload PDF (1250K) -
Noriyuki Fukunaga, Sigeki Imagawa, Takehiko Sahara, Atsushi Ishii, Mas ...1992 Volume 112 Issue 6 Pages 849-855
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADP+-dependent isocitrate dehydrogenase [IDH: EC 1. 1. 1. 42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80, 000 with a pI of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to IDH-II and -I of Vibrio ABE-1) showed an immunochemical distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35%, β-pleated sheet in the native state.View full abstractDownload PDF (817K) -
Noriko Yamaguchi, Shioko Kimura, O. Wesley McBride, Hisae Hori, Yoshih ...1992 Volume 112 Issue 6 Pages 856-863
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an effort to identify new members of the collagen family, we screened a human placenta cDNA library with a collagenous probe. A novel 3.7 kb cDNA was identified encoding an open reading frame of 1, 186 amino acids and containing a termination codon. The predicted polypeptide consists of 9 repetitive collagenous (stretches of Gly-X-Y) and several noncollagenous segments. Two cysteinyl residues separated by two amino acid residues (Cys-X-X-Cys) are regularly located in the N-terminal region of each non-collagenous segment. The deduced amino acid sequence described above is distinct from those of known types of collagen. Therefore, this novel collagen chain is designated α1(XVI). Northern blot analysis revealed an α1(XVI) mRNA of 5.2 kb, indicating that the overlapping cDNA clones isolated in this study covered nearly three-fourths of the mRNA. As a tool for further study on the expression of type XVI collagen, we prepared an antibody against the nonadecapeptide CFLSLERPRAEEARGDNSE, derived from the putative translation product of the cDNA. In immunoblot analysis, the antibody recognized a 160 kDa protein, which was bacterial collagenase-sensitive. Immunohistochemical stainings of human placental tissues with anti-peptide antibody revealed a positive reaction with amnion, the membranous tissue lining the amniotic cavity. The gene of α1(XVI) chain, COL16A1, is mapped on the short arm of human chromosome 1 (1p13-p34).View full abstractDownload PDF (3358K)
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