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Volume 115, Issue 1
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Hirota Fujiki, Masami Suganuma1994 Volume 115 Issue 1 Pages 1-5
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOkadaic acid is a potent tumor promoter on mouse skin and in rat glandular stomach, and an inhibitor of PP-1 and PP-2A. How okadaic acid biochemically induces tumor promotion in these tissues was reviewed. Okadaic acid bound to a catalytic subunit of PP-1 and PP-2A and induced hyperphosphorylation of proteins, such as vimentin, cytokeratins, HSP 27, and tumor suppressor gene products. Since one of the okadaic acid class compounds, microcystin-LR, induced tumor promotion in rat liver, the okadaic acid pathway mediated through inhibition of PP-1 and PP-2A is seen to be a general biochemical process of tumor promotion in various organs. The biochemical mimicry of okadaic acid by TNF-α led us to find that TNF-α is an endogenous tumor promoter. The study of tumor promotion in two-stage carcinogenesis experiments with the okadaic acid class of compounds engender-ed a new tumor promoter applicable to human cancer development.View full abstractDownload PDF (573K) -
Masatomo Maeda1994 Volume 115 Issue 1 Pages 6-14
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProgress in molecular biological studies on the gastric proton pump (H+/K+-ATPase) now enable us to discuss not only its subunit protein structures and catalysis but also the organizations of its subunit genes and their cell-specific transcription. The primary structures of the catalytic α and glycosylated β subunits are similar to those of the corresponding subunits of Na+/K+-ATPase. The residues located in the catalytic and cation binding sites have been proposed from the combined results of protein chemical studies and sequence comparisons of P-type cation transporting ATPases. Most of the positions of exon/ intron boundaries of the genes for the H+/K+- and Na+/K+-ATPase α and β subunits are conserved, suggesting that the a and β subunit genes, respectively, of the two ATPases were derived from common ancestors. In contrast to the Na+/K+-ATPase subunits, the H+/ K+-ATPase α and β subunits are expressed specifically in gastric parietal cells. Consistent with their cell-specific transcription, a gastric mucosal nuclear protein(s) was shown to recognize a sequence motif in the 5'-upstream regions of the H+/K+-ATPase α and β subunit genes. Furthermore, novel zinc finger proteins (GATA-GT1 and GATA-GT2) that bind to this motif were found in the gastric parietal cells. These proteins are likely to play important roles in transcriptional regulation of the gastric proton pump genes.View full abstractDownload PDF (1954K) -
Yoichi Hayakawa1994 Volume 115 Issue 1 Pages 15-17
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGrowth-blocking peptide (GBP) is a peptide which was initially isolated from the plasma of last instar larvae of the insect armyworm, Pseudaletia separata, parasitized by the parasitoid wasp, Cotesia kariyai. A standard GBP acid, a free carboxyl terminal GBP, was synthesized by solid phase methods, using 7-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) amino acids, respectively. The peptide synthesized using Boc amino acids gave the expected structure of GBP, while approximately 90% of the peptide synthesized using Fmoc amino acids was a peptide with a β-aspartyl (Asp) bond at position 16. The peptide containing the β-Asp bond does not exhibit any physiological effect on armyworm larvae.View full abstractDownload PDF (250K) -
Katsuhiko Sakurada, Toshihiko Ikuhara, Minoru Seto, Yasuharu Sasaki1994 Volume 115 Issue 1 Pages 18-21
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn antibody with specificity for the 20 kDa myosin light chain of smooth muscle phospho-rylated by myosin light chain kinase was developed. The antibody was raised against the phosphorylated synthetic peptide, Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-phosphoSer-Asn-Val-Phe-Cys (residues 11-22 of the myosin light chain). This antibody only recognized the monophosphorylated myosin light chain at serine 19, i.e., with no detectable recognition of nonphosphorylated or diphosphorylated serine 19 or threonine 18. The specificity was tested by EIA assaying of myosin light chain kinase activity using a 96-well plate coated with the light chain. This EIA system was as sensitive as the radioisotopic method, and the newly developed non-radioisotopic method.View full abstractDownload PDF (1414K) -
Atsuko Nagai, Daisaku Ohta1994 Volume 115 Issue 1 Pages 22-25
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHistidinol dehydrogenase (HDH), a Zn-metalloenzyme, produces His from histidinol through two successive oxidation reactions with NAD+ as a coenzyme. A mutation, His261→Asn, caused the complete loss of the Zn, thereby inactivating the enzyme, without significant structural perturbation. The ability to oxidize an intermediate, histidinalde-hyde, was restored to about 4% of that of the wild-type enzyme by adding 0.5mM MnCl2, whereas the histidinol oxidation activity could not be recovered with the metal addition. We concluded that the His residue at position 261 is essential for the ligation of the Zn of cabbage HDH.View full abstractDownload PDF (1577K) -
Spontaneous Fragmentation of Dimyristoylphosphatidylcholine Vesicles into a Discoidal Form at Low pHHyun-Sook Han, Hyoungman Kim1994 Volume 115 Issue 1 Pages 26-31
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon prolonged incubation at low pH, the turbidity of a dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicle (SUV) suspension underwent an initial increase follow-ed by a decrease to below the starting value. Electron microscopic observation of the vesicle suspension, after the turbidity minimum was reached, revealed the formation of discoidal particles which were smaller than the original vesicles. NMR studies indicated that all the choline groups in the discoids are accessible to added chemical shift agent, corroborating the fragmentation of the vesicles. Formation of the discoids from DMPC multilamellar vesicle (MLV) was also observed but without going through the initial increase in turbidity. The DMPC was subsequently found to be hydrolyzed into lysophosphatidylcholine (lysoPC) and myristic acid under the same conditions that caused the fragmentation. That the discoidal fragment consisted of DMPC and its hydrolysis products, lysoPC and myristic acid, was verified by electron microscopic observation of discoids when these components were mixed at neutral pH. A 50% cholesterol content prevented the fragmentation of DMPC vesicles. A model for this discoidal complex, in which a patch of DMPC bilayer is surrounded at its periphery by lysoPC and myristic acid, is proposed.View full abstractDownload PDF (2371K) -
Valentina Ruiz-Gutierrez, Carmen Maria Vazquez, Francisco Javier Quint ...1994 Volume 115 Issue 1 Pages 32-36
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn this study we examined the effect of 50% distal small bowel resection (DSBR) on the lipid composition of rat colonic mucosa, including the fatty acid composition of major phospho-lipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidyl-inositol (PI), lysophosphatidylcholine (LPC), and sphingomyelin (SM). Phospholipid, free cholesterol, triacylglycerols, diacylglycerols, and cholesterol ester levels were not affected by the surgical operation. PE together with PC accounted for more than 60% of total phospholipid, in both groups of animals. The relative percentages of PE, PC, PI, SM, and cardiolipin (CL) remained unaffected by DSBR. However, a decrease in the content of LPC was observed in 50%-resected animals. Different fatty acid composition changes in the colonic phospholipid classes were observed after resection. PC fraction contained the highest amounts of saturated fatty acids, but monounsaturated fatty acids were present in high levels in PI fraction, in both groups of animals. DSBR produces different changes in the levels of linoleic and arachidonic acids. These results suggest that the lipid composition and the fatty acid profile of the different phospholipid fractions change in the adaptive response of colon to intestinal resection.View full abstractDownload PDF (589K) -
Hideo Maeda, Kazuyo Kasahara1994 Volume 115 Issue 1 Pages 37-40
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe genetically obese Zucker rat is a well-characterized model of early-onset human obesity. The 120 kDa protein was recently found in the liver cytosol of obese Zucker rats at levels higher than that in lean Zucker rats. We isolated this protein using precipitation with ammonium sulfate, DEAE-Sephacel chromatography, and preparative polyacrylamide gel electrophoresis; the product showed a single band on SDS-polyacrylamide gel electropho-resis. Immunoblotting analysis revealed that the 120 kDa protein was predominantly localized in the liver cytosol of obese Zucker rats. The amount of this protein in lean Zucker rats was less than one-fifth of that found in obese Zucker rats. Further, there were only trace amounts of this protein in the lung tissues, and no detectable amount in other tissues, such as kidney, epididymal adipose tissue, brain, spleen, skeletal muscle, or serum, in either strain of rat. These data suggest that the 120 kDa protein contributes to the abnormal lipid metabolism in obese Zucker rats.View full abstractDownload PDF (2682K) -
Phadungchom McClelland, Leonard P. Adam, David R. Hathaway1994 Volume 115 Issue 1 Pages 41-46
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+-dependent protease, calpain II, isolated from vascular smooth muscle was found to be a substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in vitro. Phosphorylation was dependent upon prior autolysis of the regulatory subunit of calpain II. The stoichiometry of phosphorylation of native, unautolyzed calpain II was 0.02±0.01 mol PO4 /mol enzyme while for autolyzed calpain, the stoichiometry was 1.04±0.15 mol PO4 /mol enzyme. All phosphate was incorporated into the 76 kDa catalytic subunit of calpain II. A single serine residue in domain III of the catalytic subunit was identified as the phosphate acceptor: RGS*TAGGCR. Phosphorylation doubled enzyme activity mea-sured both as proteolysis of an exogenous substrate (α-casein) as well as by intermolecular catalytic subunit autolysis. The effects of phosphorylation could be reversed by dephos-phorylation using a type IIA phosphoprotein phosphatase. These results demonstrate that calpain II possesses a latent CaM kinase II phosphorylation site that is unmasked by autolysis.View full abstractDownload PDF (3509K) -
Naohiro Yoshigi, Yukio Okada, Hirohisa Sahara, Shohei Koshino1994 Volume 115 Issue 1 Pages 47-51
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolymerase chain reaction (PCR) amplification of mRNA from developing barley (cultivar Haruna two-rows) endosperm was used to clone and sequence full-length cDNA encoding, β-amylase. The, β-amylase cDNA was 1, 775 by in length. The β-amylase was deduced to be composed of 535 amino acid residues and its molecular weight was calculated to be 59, 610. Kreis et al. reported that the β-amylase cDNA from barley (cultivar Hiproly) was 1, 754 bP in length and coded for a polypeptide of 535 amino acids [Eur. J. Biochem. (1987) 169, 517-525]. A comparison of the β-amylase sequences from Haruna two-rows and Hiproly barleys revealed nine differences in the nucleotide sequence which resulted in three changes in the amino acid residues and 21 additional nucleotides at its 3'-end in the cultivar Haruna two-rows. The three changes were as follows: Ala-233, Ser-347, Met-527 (Haruna two-rows) and Val-233, Met-347, Ile-527 (Hiproly). Lundgard and Svensson pointed out that 23 amino acid residues of the peptide fragment derived from the COOH-terminal region of barley (cultivar Gula) β-amylase were in agreement with the deduced amino acid sequence reported by Kreis et al., with the exception of a single position (Met-527 compared to Ile) [ Carlsberg Res. Commun. (1986) 51, 487-491]. Our findings described above showed Met-527 is reasonable. In the cases of β-amylases from soybean and sweet potato, the positions that corresponded to those at 233 and 347 in the amino acid sequence of β-amylase from barley were Ala and Ser, respectively. Therefore, Ala-233 and Ser-347 in the amino acid sequence of barley .β-amylase were thought to be reasonable. Sequence homology of barley β-amylase with the enzymes from soybean and sweet potato amounted to 66.7 and 59.2%, respectively.View full abstractDownload PDF (604K) -
Yoshito Abe, Tadashi Ueda, Taiji Imoto1994 Volume 115 Issue 1 Pages 52-57
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe developed a method for reducing disulfide bonds in proteins under weakly acidic conditions by use of 2-aminothiophenol. The disulfide bonds in hen egg-white lysozyme, ribonuclease A, and soybean trypsin inhibitor were quantitatively reduced by 2-amino-thiophenol in phosphate buffer, pH 6, containing 8M Gdn HCI, 1mM EDTA, and 20% ethanol, for 60 min at 40°C. On analysis of the RP-HPLC patterns of tryptic peptides, which were derived from reduced and S-alkylated lysozyme and ribonuclease A at pH 6, it was confirmed that no side reaction occurred. Moreover, the reduction under weakly acidic conditions was demonstrated to be applicable for the location of such a labile residue as O-acetylated tyrosine.View full abstractDownload PDF (598K) -
Kuniko Akama, Shusaku Oka, Tohru Tobita, Hiroaki Hayashi1994 Volume 115 Issue 1 Pages 58-65
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe boar transition protein 3 (TP3) was extracted with acid from the isolated late spermatid nuclei or directly from the testes, fractionated with trichloroacetic acid, and reduced and carboxymethylated (RCM-). RCM-TP3 from the nuclei was purified by HPLCs on Nucleosil 300 7C18 and Hitachi #3057, and that from the testes, by ion-exchange chromatography on CM-Sephadex C-25 and HPLCs on Nucleosil 300 7C18 and Chemcosorb 7C8. The two TP3 preparations were identical in acid-urea- and SDS-gel electrophoretic mobilities and amino acid composition. The primary structure of TP3 was determined by manual Edman degradation of the peptides obtained by lysyl endopeptidase-digestion or by α-chymotryp-sin-digestion of RCM-TP3 from the testes, and by automated Edman degradation of it. Boar TP3 is a basic protein of 76 residues: H-AKVTEKSWQPQTTSTKRWKKRKTPSQPRSR-GKVRKIYKKVKRPLHVCSRKKYSPKVITTSRRQKRARRANKFETIP-OH, and it shows 27% homology with boar TP1. TP3 is composed of an N-terminal region (1-19) having two characteristic tryptophan residues (8 and 18) which is absent in the known TPI group, and a C-terminal region (20-76) having a close resemblance to boar TP1.View full abstractDownload PDF (3844K) -
Yoshinori Harada, Toshiya Senda, Takeshi Sakamoto, Kazunori Takamoto, ...1994 Volume 115 Issue 1 Pages 66-75
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSApoproteins, having various molecular weights, of octopus rhodopsin (oRh, 455 amino acids), which is a typical transmembrane protein, were expressed in Escherichia coli with an inducible expression system using promoter φ10 of the T7 phage. Fifteen synthetic genes (212-1, 365 bp) for oRh (1) were cloned downstream from gene 10 of the T7 phage (846 bp), under the control of promoter Φ10. An expression vector for mature oRh containing no extra peptide resulting from gene 10 was also constructed. Protein productivities were mainly evaluated by ELISA using monoclonal antibodies. The expression level in E. coli of the fused oRh genes varied between 1 and 200mg/liter of the culture medium (from approxi-mately 0.25 to 50% of the total cell protein, respectively), depending on the fused oRh genes. The amount of mature oRh protein expressed in E. coli was approximately 0.1 to 1mg/liter. Hydropathy index analysis of gene products showed a significant negative correlation (Ρ=-0.63) between expression level of oRh gene products in E. coli and their hydrophobic characteristics. Wavelength shifting of the absorption maximum by exogenous addition of retinal to apoprotein similar to that of authentic oRh was demonstrated in the membrane fraction of E. coli expressing mature opsin.View full abstractDownload PDF (3255K) -
Tomofumi Inokuchi, Ken-ichiro Kobayashi, Shiro Horiuchi1994 Volume 115 Issue 1 Pages 76-81
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid proteinase different from pepsin was purified from bullfrog (Rana catesbeiana) gastric mucosa by chromatography on hydroxyapatite, Q-Sepharose, Con A-Sepharose 4B, and Mono Q columns. Its molecular weight after purification was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 45 kDa under reducing conditions and about 90 kDa under nonreducing conditions. Thus, it is a dieter of two identical subunits. On acid treatment, the molecular weight of the subunit decreased from 45 to 42 kDa, showing a similar change to that of pepsinogen in its activation under acidic conditions. Therefore, the enzyme was thought to have both a proform and a mature form. It preferred hemoglobin to other protein substrates examined and showed broad optimal activity in the range of pH 2.0 to 3.5 towards hemoglobin. Its proteolytic activity, like that of porcine pepsin, was strongly inhibited by pepstatin. Its amino acid composition was similar to those of other aspartic proteinases. From these results, the enzyme was identified as a cathepsin E type acid proteinase of bullfrog, and cathepsin E type enzyme was purified from anuran for the first time.View full abstractDownload PDF (1517K) -
So Young Lee, Hyun Joo Moon, Shoichiro Kurata, Takeshi Kurama, Shunji ...1994 Volume 115 Issue 1 Pages 82-86
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInjection of Escherichia coli into larvae of the coleopteran Holotrichia diomphalia results in the appearance of antibacterial activity in the hemolymph. An antibacterial protein, named holotricin 2, was purified from larvae of this insect and characterized. A eDNA clone for holotricin 2 was isolated and its complete sequence was determined. This protein was found to inhibit the growth of Gram-negative bacteria and to consist of 72-amino acid residues with no cysteine residues. Its amino acid sequence is similar to that of coleopter-icine, an antibacterial protein isolated from larvae of the coleopteran Zophobas atratus.View full abstractDownload PDF (1634K) -
Miho Suzuki, Hideaki Mizuno, Yasuo Akanuma, Hiroshi Akanuma1994 Volume 115 Issue 1 Pages 87-92
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA pyranoid polyol, 1, 5-anhydroglucitol (AG), generally occurs in the human body as a humoral component. The plasma AG concentration in healthy individuals is maintained at a constant level, but it is markedly decreased in diabetes mellitus. This is due to hyperglycemia-dependent abolishment of renal AG retention. Hence, the plasma AG concentration has been established as a clinical marker for duration of hyperglycemia and since 1991 it has been practically applied to diabetic care in Japan. However, the details of the metabolism of AG and its physiological significance generally remain to be studied. In this study, we confirmed AG synthesis in cultured cells of a rat hepatoma line, Reuber H-35, in which AG was found to be derived from glucose, with retention of all six carbon atoms in the pyranoid structure. The fraction of the total glucose consumed by the cells, which was converted to AG (conversion efficiency) was at most 5×10-6. The conversion efficiency increased at higher glucose concentrations (mM orders) where the glucose consumption rate was saturated. Since the rate of the hexokinase reaction, one of the rate-limiting steps in glucose consumption, has been estimated to be saturated at μM orders of glucose concentra-tion, this observation was interpreted as indicating that AG is synthesized through a pathway which does not share the hexokinase reaction with glucose utilization. The presence of precursors other than glucose was also indicated in the time-course study of AG synthesis. Further, the amount of AG synthesized daily in humans is significant in comparison with the amount obtained from the diet.View full abstractDownload PDF (707K) -
Dong-Woon Kim, Tohru Yoshimura, Nobuyoshi Esaki, Etsuko Satoh, Kenji S ...1994 Volume 115 Issue 1 Pages 93-97
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(β-aminoethyl)cysteine (SAEC), S-(β-aminopropyl)cysteine (SAPC), and S-(β-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorb-ance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that ω-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 106-108 times more active than the K239C mutant enzyme. These results suggest that ω-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.View full abstractDownload PDF (619K) -
Jin-Yan Cui, Kuniaki Mukai, Kazuhiko Saeki, Hiroshi Matsubara1994 Volume 115 Issue 1 Pages 98-107
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESScDNA clones encoding subunits I, II, and IX of Euglena gracilis mitochondrial complex III have been isolated from a λ gtll cDNA expression library by immunoscreening with an antiserum against the complex of the organism. Determination of the nucleotide sequences and amino-terminal amino acid sequences of purified subunits revealed that subunits II and IX, respectively, consist of 432 and 70 amino acids as their mature forms and possess potential presequences of 42 and 30 amino acids. The amino-terminal parts of the presequences had typical structural features of the mitochondrial targeting signal. Such features were also found at the amino-terminal region of the predicted subunit I protein, which comprises 494 residues. However, the amino terminus of the purified subunit I could not be detected, possibly because of a post-translational modification. Euglena subunits I and II both showed similarities to the members of the protein family which comprises complex III core proteins, mitochondrial processing peptidases (MPP) and processing enhancing proteins (PEP). Namely, the Euglena subunit I could be assigned to core 1 protein and the subunit II to core 2 protein in the family. In contrast, the subunit IX seemed to be peculiar to Euglena complex III. At 5'-untranslated regions, the three cloned cDNAs for subunits I, II, and IX had a common poly(T)CG structure which has also been reported for other Euglena cDNAs of nuclear genes.View full abstractDownload PDF (1112K) -
Yutaka Matsushima, Dong-Woon Kim, Tohru Yoshimura, Seiki Kuramitsu, Hi ...1994 Volume 115 Issue 1 Pages 108-112
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active-site lysine residue of thermostable aspartate aminotransferase, Lys-239, to which the cofactor, pyridoxal 5'-phosphate (PLP), is bound, has been converted to Cys by site-directed mutagenesis. The thiol group of Cys-239 was chemically aminoethylated with ethylenimine. Amino acid analysis of the modified enzyme showed that it contained about 1 mol of S-(2-aminoethyl)cysteine (SAEC) per mol subunit. The activity of the mutant enzyme (K239SAEC) was about 14% of that of the wild-type enzyme. No significant difference in thermostability was found between the wild-type and K239SAEC enzymes. The UV-visible spectrum of K239SAEC showed a peak (λmax 380 nm), due to absorption by the cofactor, at a 20 nm longer wavelength than that of the wild-type enzyme. The circular dichroism band due to the bound cofactor of K239SAEC also shifted toward a 20 nm longer wavelength. We determined kinetic parameters (rate constants, kmax, and dissociation constants, Kd, for the substrates) for each half transamination catalyzed by the wild-type and K239SAEC mutant enzymes by the stopped-flow method. The kmax values for the mutant enzyme reactions were 2.6-24 times lower than those for the wild-type enzyme ones. The two enzymes showed similar Kd values for the same substrates except glutamate; the mutant enzyme showed higher affinity for glutamate than the wild-type enzyme.View full abstractDownload PDF (598K) -
Naokazu Sasagasako, Takuro Kobayashi, Yukiko Yamaguchi, Nobue Shinnoh, ...1994 Volume 115 Issue 1 Pages 113-119
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe metabolism of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) was studied using cultured fibroblasts deficient in acid β-glucosidase activity. In fibroblasts from patients with Gaucher's disease, in vitro β-glucosidase activities were 2.7-11.7% and 4.8-13.6% of control values when 4-methylumbelliferyl β-D-glucoside and GlcSph were used as substrates, respectively. In spite of the enzyme deficiency, GlcCer and GlcSph, the natural substrates of the deficient enzyme, did not accumulate in the cells. When normal fibroblasts were incubated with conduritol B epoxide (CBE), a specific inhibitor of acid β-glucosidase, the in vitro enzyme activities decreased dose-dependently (2.2-2.4% of control values at 50 μM CBE), and GlcCer and GlcSph accumulated in the cells at concentra-tions of CBE higher than 50 μM. To investigate the intracellular metabolism of GlcCer and GlcSph, either radioactive GlcCer or GlcSph was loaded onto cultured fibroblasts. In fibroblasts treated with a high dose of CBE (1mM), the degradation of GlcCer and GlcSph was retarded (5-21% on day 7; normal range, 81-99%), while in fibroblasts from patients with Gaucher's disease, both the pattern and rate of the degradation of the lipids (83-97% on day 7) were almost the same as those seen in the control cells. These results indicate that in Gaucher's disease fibroblasts the intracellular metabolism of GlcCer and GlcSph is normal in spite of the deficiency in β-glucosidase activity. At present it is not known why there is a discrepancy between the in vitro enzyme activity and the intracellular metabo-lism of the substrates, but the residual activity in the fibroblasts may well play a critical role in the in vivo degradation of the lipids in the fibroblasts, and these data may therefore explain the fact that the accumulation of GlcCer and GlcSph is confined to certain tissues in patients with Gaucher's disease.View full abstractDownload PDF (3181K) -
Alan Y. Deng, Louis L. Martin, Joseph L. Balwierczak, Arco Y. Jeng1994 Volume 115 Issue 1 Pages 120-125
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA soluble protease which effectively inactivated endothelia-1 was purified 2, 700-fold from rat kidney using DE52 anion exchange, concanavalin A-Sepharose, thiopropyl-Sepharose, and Mono P column chromatography. The overall recovery of enzyme activity was 12%. This enzyme appeared to contain two subunits with molecular weights of 34 and 21 kDa. The molecular weight of the active enzyme complex was estimated to be 82 kDa by gel filtration. It was shown to have a pI between 4.8 and 5.2 and a pH optimum of 5.5. Its activity was inhibited by benzyloxycarbonyl-Phe-AlaCHN2 and p-hydroxymercuribenzoic acid, thiol protease inhibitors, and by phenylmethylsulfonyl fluoride, a serine protease in-hibitor, but not by metalloprotease inhibitors or other serine protease inhibitors. The purified enzyme degraded endothelin-1 rapidly; KM and Vmax values were 5.9 μM and 0.40 μmol/mg/min, respectively. It removed the carboxyl terminal tryptophan of endothelia-1 by specifically cleaving the Ile20-Trp21 peptide bond, yielding a peptide which was three orders of magnitude less potent than endothelia-1 in causing contraction of porcine coronary arterial rings. In contrast, proendothelin-1 was not degraded by this enzyme. These results are consistent with published findings that show the clearance rate for proendothelin-1 in the pig to be significantly slower than that for endothelin-1. Our study suggests that this enzyme may play a role in the homeostasis of circulating endothelin-1 and contribute to the regulation of vascular tone.View full abstractDownload PDF (1821K) -
Kelly P. Williams, Denise N. Fujimoto, Tan Inoue1994 Volume 115 Issue 1 Pages 126-130
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo adenosine residues, universally conserved among group I introns, are located in the L4 egion of the catalytic core. Base-substitution mutations in these adenosines resulted in diminished in vitro self-splicing activity of the Tetrahymena group I intron, more severely for double than for single mutations. The defect caused by the mutation of the L4 adenosines was manifest at the first step of splicing (cleavage of the 5' splice site by a guanosine molecule), and could be overcome by increasing the magnesium ion concentration of the reaction buffer. In contrast, a related activity of the group I intron, specific hydrolysis of the 3' splice site, was virtually unaffected by the L4 adenosine mutations; this reaction must require an active conformation of the catalytic site of the ribozyme and also depends on the recognition of guanosine (the guanosine residue that precedes the 3' splice site in all group I introns). These results suggest that the role of the L4 adenosines is limited to improving the reactivity of the 5' splice site during splicing. We also found that mutations that eliminate the 5' splice site-bearing P1 stem and the P10 stem have little or no effect on specific hydrolysis of the 3' splice site.View full abstractDownload PDF (3120K) -
Kohei Hosaka, Jun-ichi Nikawa, Tsutomu Kodaki, Satoshi Yamashita1994 Volume 115 Issue 1 Pages 131-136
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA dominant mutation of Saccharomyces cerevisiae, CSE1, caused a decrease in the expression of the INO1 gene product, inositol-1-phosphate synthase. The residual activity was completely repressed by the addition of choline to the medium. A mutant carrying this mutation could not grow in the presence of choline unless inositol was added to the medium. Here we report a suppressor gene of the CSE1 mutation, SCS1(suppressor of CSE1), which was cloned by complementation of CS-E1 with a wild-type multicopy yeast genomic library. The cloned SCS1 gene contained an open reading frame which encoded 304 amino acid residues with a calculated molecular mass of 34, 234 Da, and the sequence coincided with that of the INO2 gene. An scs1/ino2 null mutant constructed by gene replacement was viable, but auxotrophic for inositol and choline, and used for determination of the mRNA levels of various phospholipid-synthesizing enzymes. In agreement with the reported data for ino2 mutants the disruptant showed decreased expression of the INO1 and PSS genes, which are known to be regulated by inositol and choline. In addition, we newly found that the disruption of SCS1/INO2 also caused a decrease in the expression of the CKI, PEM1, and PEM2 genes, which we previously showed to belong to the inositol-choline-regulated gene family. These results confirm and strengthen the conclusion that the SCS1/INO2 gene is required for expression of inositol-choline-regulated genes in phospholipid synthesis.View full abstractDownload PDF (2245K) -
Kazuo Hosoi, Susumu Tsunasawa, Kinji Kurihara, Hideyuki Aoyama, Takao ...1994 Volume 115 Issue 1 Pages 137-143
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe protein structure, kinin-releasing activity, and tissue distribution of four major proteinases of mouse submandibular gland (mK22, mK9, proteinase F, proteinase P) were studied. When compared with the deduced amino acid sequence of each member of the tissue (glandular) kallikrein gene family, the amino acid sequence of proteinase F determined (approximately 40% of the total) was found to agree completely with the deduced amino acid sequence of mKlk-1. The proteinase P sequence, on the other hand, agreed with that of the product of mKlk-13, mK13 (prorenin-converting enzyme). Proteinase F had the strongest kininogenase activity for both low-molecular-weight and high-molecular-weight kininogen, while mK22 had 1/6 and 1/50 the activity of proteinase F for the respective kininogen substrate. Kininogenase activities of mK9 and proteinase P were less than 1/100 of the activity of proteinase F for both substrates. Acting on the two kininogen substrates, kallikreins mK22, mK9, and proteinase F, but not proteinase P, specifically released bradykinin, suggesting that the former three kallikreins strictly recognized peptide sequences around bradykinin in these substrate molecules but proteinase P recognized several sites in these molecules. Significant amounts of proteinase F, but not mK22 and others, were present in the urine, pancreas and digestive organs, as well as in the salivary glands. The present results revealed that the former proteinase F is identical to mK1, tissue/renal kallikrein, and confirmed its characteristics as a true kallikrein on the basis of its kinin-releasing activity and tissue distribution.View full abstractDownload PDF (825K) -
Sachio Morimoto, Iwao Ohtsuki1994 Volume 115 Issue 1 Pages 144-146
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe role of troponin C in determining the characteristic differences in contraction between fast-twitch skeletal and cardiac muscles was investigated with rabbit skinned psoas fibers and left ventricular trabeculae by exchanging their troponins C using a technique involving extraction and replacement. In both muscle types, skeletal troponin C conferred higher cooperativity on the Ca2+-activated tension development than cardiac troponin C, indicat-ing that troponin C plays a crucial role in determining the cooperativity in Ca2+ activation of vertebrate striated muscle. Furthermore, the foreign type of troponin C was found to confer lower Ca2+ sensitivity on the tension development in both fast-twitch skeletal and cardiac muscles than the native type of troponin C. This indicates that the Ca2+ sensitivity of each muscle type is determined by troponin C interactions with other troponin subunits and not by troponin C alone.View full abstractDownload PDF (299K) -
Kei Maruyama, Takeshi Endo, Hideaki Kume, Yuuki Kawamura, Nobuyuki Kan ...1994 Volume 115 Issue 1 Pages 147-149
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA partial cDNA encoding 811 amino acids of connectin (titin), a giant elastic protein of muscle (3, 000 kDa), was cloned from a chicken embryonic skeletal muscle cDNA library using antibodies to muscle connectin. The encoded product was the C terminal segment of connectin. The predicted sequences consisted of 5 type II motifs (immunoglobulin C2 type) separated by 5 interdomain insertions. One interdomain insertion had significant homology (RSP) to KSP repeats found in human cardiac C-terminal connectin and another had a high sequence homology to porin (67.7%; 31 amino acids).View full abstractDownload PDF (1243K) -
Reiko Sugaya, Satoshi Ishimaru, Kaoru Saigo, Yasufumi Emori1994 Volume 115 Issue 1 Pages 150-155
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have identified a novel gene encoding a putative protein kinase from a Drosophila genomic library. The gene, about 2 kbp in length, consists of four exons and codes for a protein of 349 amino acid residues. The deduced sequence shows significant similarity to various kinases, especially to a subgroup of Ser/Thr kinases related to Cdc2 kinase; thus, the gene was termed Dcdrk (Drosophila cdc2-related kinase gene). Among the kinases examined, mammalian galactosyltransferase-associated 58 kDa protein kinase showed the highest homology (about 50% identity in the kinase domain) to Dcdrk kinase. Northern blot analysis revealed that the Dcdrk mRNA is expressed throughout development in nearly constant amounts. Moreover, a whole mount in situ hybridization experiment showed that the Dcdrk mRNA is ubiquitously distributed in almost all embryonic cells and tissues, suggesting a universal function of Dcdrk, possibly in cell cycle regulation.View full abstractDownload PDF (1694K) -
Masahiro Iwasaki, Hideyuki Hayashi, Hiroyuki Kagamiyama1994 Volume 115 Issue 1 Pages 156-161
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Schiff base formed between Lys258 of Escherichia coli aromatic amino acid amino-transferase (ArAT) and the coenzyme pyridoxal 5'-phosphate (PLP) has a pKa value of 6.65. The pH dependency of the kinetic parameters was consistent with a mechanism by which the enzymatic form with the nonprotonated Schiff base productively binds aspartate, phenylalanine, and tryptophan. The Schiff base pKa value rose by 1.7-2.1 unit on binding of substrate analogs, and this strongly suggested protonation of the Schiff base upon formation of the Michaelis complex with substrates. The protonated “internal” Schiff base in the Michaelis complex is supposed to be attacked by the deprotonated substrate amino group, and this explains excellently the mechanism of transaldimination to form the PLP-substrate Schiff base. Phenylpropionate and indolepropionate caused similar in-creases in the pKa value to maleate. [Arg292→Ala] ArAT showed the same pKa value as the wild-type enzyme. Therefore, neutralization of Arg292 by ω-carboxylate of dicarboxylic ligands, which had been well documented in aspartate aminotransferase to increase the Schiff base pKa, has little effect on the protonation of the Schiff base in ArAT. Thus the structure of ArAT is deliberately organized so that the Schiff base pKa is effectively modulated by substrates having only one carboxylate group.View full abstractDownload PDF (672K) -
Hideko Yamamoto, Yuji Mizuno, Kensuke Hayashi, Ikuya Nonaka, Mikiharu ...1994 Volume 115 Issue 1 Pages 162-167
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody MA4-2 against a dystrophin-associated protein 35DAG (A4) was established and applied to examine the distribution of 35DAG in monkey tissue and its expression in DMD patients. In immunoblotting after two-dimensional gel electrophoresis of crude skeletal muscle extracts, MA4-2 reacted exclusively with an apparent single spot located in a similar position to rabbit 35DAG in each animal examined. 35DAG was detected only in striated muscles (quadriceps femoris and cardiac muscles), but not in other tissues examined, including smooth muscle (aorta, uterus), brain, nerve, lung, and liver. This distribution pattern is the same as that of 50DAG but different from that of 43DAG (A3a) [Mizuno et al. (1993) J. Biochem. 114, 936-941]. In Duchenne muscular dystrophy muscles, 35DAG was distinctly present, but greatly reduced in amount. This is also the case with 50DAG. On the basis of these results, it is concluded that 35DAG and 50DAG are striated muscle-specific and may be important in the pathogenesis of Duchenne muscular dystrophy.View full abstractDownload PDF (2255K) -
Crystallization and Preliminary Crystallographic Data of the α-Amylase Inhibitors, Haim I and Paim IMayuko Miyagawa, Ken-ichi Fukuhara, Katsuo Katayanagi, Kaori Ishimaru, ...1994 Volume 115 Issue 1 Pages 168-170
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo proteins that act as α-amylase inhibitors, Haim I and Paim I, were crystallized and preliminary X-ray diffraction studies on them were carried out. We also sequenced Haim I prepared from Streptomyces griseosporeus YM-25 and confirmed that it is composed of 78 amino acid residues. Crystals of Haim I were grown from ammonium sulfate solution mixed with ethanol by the vapor diffusion technique. The crystals grew as hexagonal bipyramids and diffracted X-rays beyond 2.0 Å resolution. They belong to the space group P6122 (or P6522) with unit cell dimensions of a=b=36.7 Å, c=192.4 Å, and contain one molecule per asymmetric unit. Paim I, a protein of 39 amino acid residues produced by Streptomyces corchorusii, was crystallized under similar conditions to Haim I. The crystals diffracted X-rays beyond 2.5 Å. They belong to the space group P41212 (or P43212) with unit cell dimensions of a=b=65.4Å, c=96.1 Å, and contain three molecules per asymmetric unit.View full abstractDownload PDF (1365K) -
Fumie Shiraishi, Kazuhiko Yamamoto1994 Volume 115 Issue 1 Pages 171-173
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of partial removal of troponin I and C on the profiles of Ca2+-sensitive ATPase activity in rabbit skeletal myofibrils was investigated by replacing the troponin C•I• T-complex in the myofibrils with exogenously added troponin T under the same conditions as those reported previously [Shiraishi et al. (1992) J. Biochem. 111, 61-65]. During the course of the troponin T treatment, the level of the ATP hydrolysis at low Ca2+ concentra-tions was elevated and the pCa for half maximum activation increased, while the cooper-ativity decreased. These changes in the parameters of the ATPase were correlated with the extent of the troponin I removal from myofibrils.View full abstractDownload PDF (1224K)
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