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Volume 114, Issue 1
Displaying 1-26 of 26 articles from this issue
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Kazunori Tanaka, Kazuyuki Mori, Shigeki Mizuno1993 Volume 114 Issue 1 Pages 1-4
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe light protein components of silk fibroin were analyzed by Western blotting using polyclonal antibodies against two synthetic peptides that are segment of the light (L-) chain [Yamaguchi et al. (1989) J. Mol. Biol. 210, 127-139] and P 25 [Chevillard et al. (1986) Sericologia 26, 435-449], respectively, and against the whole L-chain. Both the L-chain and P 25 were present with fibroin in the lumen of the posterior silk gland and in the cocoon. The L-chain was identified as the major light component that was disulfide-linked to the fibroin heavy (H-) chain, whereas P 25 was a minor component whose association with the H-chain was suggested to be non-covalent.View full abstractDownload PDF (2288K) -
Hideki Ozawa, Masaharu Kotani, Ikuo Kawashima, Masaaki Numata, Tomoya ...1993 Volume 114 Issue 1 Pages 5-8
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe established a murine monoclonal antibody (MAb) specific for ganglioside GM4 by immunizing C3H/HeN mice with chemically synthesized GM4 adsorbed to Salmonella minnesota, followed by fusion with mouse myeloma cells. The MAb, designated as AMR10, was shown to exhibit high binding specificity, reacting only with the ganglioside GM4 used for immunization and native GM4 from human brain. We determined the distribution of GM4 in adult chicken cerebellum by means of the immunofluorescence technique with the MAb. Our study revealed that GM4 expression was associated with astrocytes in the granular layer and the white matter, but not with myelin in any layers of the chicken cerebellar cortex.View full abstractDownload PDF (1760K) -
Tomoyuki Sumizawa, Tatsuhiko Furukawa, Misako Haraguchi, Akihiko Yoshi ...1993 Volume 114 Issue 1 Pages 9-14
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPartial complementary DNA (cDNA) for thymidine phosphorylase (dThdPase) was cloned by means of a polymerase chain reaction. There was complete sequence identity between the amino acid sequence deduced from the nucleotide sequence of a clone (288 nucleotides) and the residues of platelet-derived endothelial cell growth factor (PD-ECGF). The amino acid sequence of all four peptide fragments from purified human dThdPase could be aligned with that of PD-ECGF. Our data indicate that residues 125-244 of PD-ECGF are identical to the sequence of human dThdPase. The molecular weights of human dThdPase and recombinant PD-ECGF (rPD-ECGF) that lacks 10 amino acids at the amino terminal were 55 and 52 kDa, respectively. Anti-PD-ECGF antibody recognized dThdPase, and anti-dThdPase antibody recognized rPD-ECGF. rPD-ECGF had dThdPase activity and its specific activity was similar to that of purified human dThdPase. dThdPase activity and molecules were detected in COS cells transfected with human PD-ECGF cDNA, but not in nontransfected cells. The sizes of PD-ECGF and dThdPase in the transfected COS cells were identical. These data suggest that human dThdPase is identical to PD-ECGF.View full abstractDownload PDF (2669K) -
Hiroyuki Ida, Owen M. Rennert, Yoshikatsu Eto, Wai-Yee Chan1993 Volume 114 Issue 1 Pages 15-20
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA encoding human acid sphingomyelinase was initially obtained by screening a placental cDNA library in λ gtll with a synthetic oligonucleotide probe and subsequently with partial cDNA. The full-length cDNA, hPSM 55, comprised 2, 376 nucleotides, with a 5' untranslated sequence of 122 nucleotides, an open reading frame of 1, 884 nucleotides encoding a protein of 627 amino acids, and a 3' untranslated region of 370 bases. hPSM 55 was almost identical to pASM-1 FL reported by Schuchman et al. (J. Biol. Chem. 266, 8531-8539, 1991) except for a 6 base pair deletion in the signal peptide, which indicated the possible removal of valine and leucine residues between positions 36 and 37, and a463T- to C-transition, which indicated a possible substitution of155arginine for cystine. This cDNA was expressed in both COS-7 cells and Chinese hamster ovary cells. There was no increase in acid sphingomyelinase activity in either cell line following transfection. However, the correction of a single base change, 463C to T, in hPSM 55 caused increased acid sphin-gomyelinase activity in transfectants. These results suggest that the mutation of nucleotide 463C to T plays an important role in the catalytic activity of acid sphingomyelinase.View full abstractDownload PDF (1667K) -
Hideaki Mizuno, Hiroshi Akanuma1993 Volume 114 Issue 1 Pages 21-27
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe found that the glucitol concentration was extraordinarily high in bovine fetal serum, which is routinely used for cell culture in laboratories: it was as much as two orders of magnitude higher than that reported for human adult serum. We also confirmed that the serum glucitol concentration in new born babies was on average 5.5-fold higher than in the maternal serum. These observations raise the possibility that some tissue (s) demand extracellular glucitol during embryogenesis and this indicates that the cells in such tissues could be permeable to glucitol. Since the hepatic metabolism of glucitol had been reported, we investigated glucitol permeability and its metabolism in rat hepatoma cells, Reuber H-35. The cells rapidly incorporated glucitol but the mode of incorporation was unusual: the incorporation rate was still proportional to the ambient glucitol concentration at 100mM. The major part of the incorporated glucitol underwent metabolic conversion to probably negatively charged metabolites. Active synthesis of glucitol was also observed in the same cells. The cells proliferated normally in medium containing glucitol instead of glucose. This observation may indicate that glucitol can substitute for glucose in the culture of H-35 cells.View full abstractDownload PDF (844K) -
Kazuhiro Oiwa, Toshimitsu Kawakami, Haruo Sugi1993 Volume 114 Issue 1 Pages 28-32
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo obtain information about the mechanism of ATP-dependent actin-myosin sliding responsible for muscle contraction, we studied the “unitary” distance of sliding between a myosin-coated glass microneedle and actin filament arrays (actin cables) in a giant algal cell induced by iontophoretic application of ATP, attention being focused on the minimum distance of ATP-induced sliding when the amount of applied ATP was gradually decreased in the presence of hexokinase and D-glucose. The number of myosin heads interacting with actin cables was reduced to less than 100, as judged from the maximal force P0 (-100 pN) generated by myosin heads on the needle in the presence of 2mM ATP. When the amount of iontophoretically applied ATP was decreased by reducing the amount of charge passed through the ATP electrode from 80 to 2 nC, the distance of ATP-induced actin-myosin sliding decreased almost linearly from -100 to -10 nm, no detectable actin-myosin sliding being observed with further reduction of the charge passed through the electrode. The amount of external load exerted by the bent microneedle was less than 1% of Po for the sliding distance<50 nm. The actin-myosin sliding distances with a small amount of ATP slightly above the amount required to induce the minimum sliding distance were distributed around integral multiples of 10 nm, suggesting that the unitary distance of actin-myosin sliding coupled with ATP hydrolysis is of the order of 10 nm.View full abstractDownload PDF (597K) -
Rei Kikuchi-Yanoshita, Ryohei Yanoshita, Ichiro Kudo, Hiroyuki Arai, T ...1993 Volume 114 Issue 1 Pages 33-38
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhospholipid-hydrolyzing activities were examined in rat hearts with ischemia induced by occlusion of the left main coronary artery. When homogenates of ischemic heart were incubated in vitro at 37°C, a significant amount of phosphatidylethanolamine (PE) was degraded, whereas the contents of other phospholipids did not change significantly. During the incubation, a stoichiometrical amount of lysoPE was concomitantly formed. The lysoPE formed had mainly saturated fatty acids and its composition resembled that of fatty acids detected at the sn-1 position in the glycerol backbone of heart PE. No appreciable PE degradation was observed in homogenates prepared from nonischemic rat heart. No difference in phospholipase activities was found between ischemic and nonischemic heart homogenates when exogenous radioactive phospholipids were used as substrates. Rabbit anti-rat 14-kDa type II phospholipase At2 antibody suppressed the degradation of PE observed in ischemic heart homogenates. These findings indicate that the type II phos-pholipase A2 activity may be involved in the breakdown of endogenous PE in ischemic heart homogenates.View full abstractDownload PDF (671K) -
Takeshi Ikeda, Shigeru Yamaguchi, Hirokazu Hotani1993 Volume 114 Issue 1 Pages 39-44
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacterial flagellum consists of a basal body, a hook, HAP1 (hook-associated protein 1), HAP3, a long helical filament, and a cap (composed of HAP2), all connected in series. The mutant deficient in the HAP2 structural gene (fliD) of Salmonella typhimurium has flagella composed of only hook-HAP1-HAP3 and excretes flagellin monomers into the culture medium. However, when purified HAP2 was added to this mutant, the flagellin stopped leaking out and flagellar filaments grew. Turnover of HAP2 was not necessary for the growth of a filament. Therefore HAP2 facilitates the polymerization of endogenous flagellin, apparently without falling off the filament tip. This experimental system with exogenous HAP2 allowed us to synchronize filament growth; the average rate of filament growth can be estimated by measuring the length of grown filaments at various time periods in electron micrographs. The initial growth rate was about 30 nm/min, which corresponds to one flagellin per second.View full abstractDownload PDF (2800K) -
Shigeo Yamamoto, Satoko Nagata, Kaoru Kusaba1993 Volume 114 Issue 1 Pages 45-49
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHomospermidine synthase, catalyzing the formation of homospermidine [H2N (CH2)4NH-(CH2)4NH2] from putrescine and NAD+ with concomitant liberation of NH3, was purified 600-fold over the crude extract with a yield of about 14% to homogeneity from Acineto-bacter tartarogenes ATCC 31105. The enzyme had a native molecular mass of 102 kDa, with a pI of 5.0, and was apparently composed of two identical subunits (52 kDa), suggesting that a single protein catalyzes two serial reactions, oxidation of putrescine to 4-aminobutyral-dehyde and subsequent reduction of the putative Schiff base formed between this aldehyde and a second molecule of putrescine to homospermidine. The Km. values for putrescine and NAD+ were 280 and 18 μM, respectively. 1, 3-Diaminopropane and cadaverine were inactive as substrates, and NAD+ could not be replaced by NADP+. 1, 3-Diaminopropane and NADH were potent competitive inhibitors. The enzyme had a pH optimum of 8.7, was most active at 30°C, and required K+ and dithiothreitol for full activity. Putrescine and NAD+ protected the enzyme from the inhibition by thiol reagents. The NH+-terminal amino acid sequence was AQWPVYGKISGPVVI. Some of these properties were compared with those of the homospermidine synthases from a photosynthetic bacterium, Rhodopseudomonas viridis and a plant, Lathyrus sativus.View full abstractDownload PDF (1271K) -
Tang Zhu, Shu-ichi Matsuzawa, Yusuke Mizuno, Kunimi Kikuchi1993 Volume 114 Issue 1 Pages 50-54
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSActivities of protein phosphatases PP1 and PP2A were determined in T and B lymphocytes of autoimmune-prone MRL/MpJ-lpr/lpr mice (MRL/Ipr mice) and two control strains, MRL/MpJ- +/+ mice (MRL/+/+mice) and C3H/HeJ mice. Potential PP1 activity, which was measured after treatment of cell extract with Co2+-trypsin, was much higher in T lymphocytes than B lymphocytes. However, no difference in the activity was observed between MRL/lpr mice and the controls. Spontaneous PP2A activity showed similar levels in T and B lymphocytes from normal mice, but potential PP2A activity, which was measured after treatment with 2-mercaptoethanol, was significantly higher in T lympho-cytes from MRL/lpr mice than those from controls. No differences were detected in PP1 or PP2A activities in B lymphocytes. From these results, our previous data [Matsuzawa, S. et al. (1992) J. Biochem. 111, 472-477] demonstrating increases in potential activities of PP1 and PP2A in lymphoid tissues from autoimmune MRL/lpr mice can be interpreted as follows. 1) The increase in potential PP1 activity of the lymphoid tissues from MRL/lpr mice is caused by replacement of B lymphocytes by abnormal T lymphocytes, which accumulate in enormous numbers. 2) The increase of potential PP2A activity in the lymphoid tissues from MRL/lpr mice is caused by the increase in this activity in their T lymphocytes.View full abstractDownload PDF (511K) -
Nobuaki Adachi, Takeo Kubo, Shunji Natori1993 Volume 114 Issue 1 Pages 55-60
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we purified a DNA binding protein from a nuclear extract of the fat body of Sarcophaga peregrina larvae that binds to the ACCACAACA motif in the 5'-upstream region of the arylphorin gene, and suggested that this protein is a transcriptional activator of the arylphorin gene. In this study, we detected and purified the same protein (ABP-1) from an embryonic cell line of Sarcophaga that does not express the arylphorin gene. Unlike the fat body, which synthesizes arylphorin actively, the embryonic cells were found to contain an additional DNA binding protein (ABP-2) that bound to the same DNA probe as ABP-1, suggesting a novel mechanism of regulation of the arylphorin gene.View full abstractDownload PDF (2821K) -
Yoshihiro Konishi, Katsuyuki Tanizawa, Susumu Muroya, Toshio Fukui1993 Volume 114 Issue 1 Pages 61-68
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2. 7. 7. 9], which catalyzes the reversible uridylyl transfer between glucose 1-phosphate and MgUTP, has been cloned by the use of oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1, 689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant sequence similarities with the enzymes from potato tuber and a slime mold, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the purified enzyme was incubated with an affinity labeling reagent, uridine triphosphopyridoxal, and then reduced with sodium borohydride. The enzyme was inactivated rapidly and irreversibly by the reagent at low concentrations. The inactivation was almost completely retarded by UDP-glucose and MgUTP. Structural analysis of the labeled enzyme revealed that three lysyl residues, Lys291, Lys357, and Lys396, were modified by the reagent. The three lysyl residues are conserved at the corresponding positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the active-site structure of bovine liver UDP-glucose pyrophosphorylase is very similar to that of the potato tuber enzyme.View full abstractDownload PDF (952K) -
Kunishige Kataoka, Harumi Takada, Tohru Yoshimura, Setsuo Furuyoshi, N ...1993 Volume 114 Issue 1 Pages 69-75
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhenylalanine dehydrogenase from Thermoactinomyces intermedius and leucine dehy-drogenase from Bacillus stearothermophilus show a 59% sequence similarity in their substrate-binding domains, although their substrate specificities are different. We prepared a phenylalanine dehydrogenase mutant enzyme whose inherent hexapeptide segment (124F-V-H-A-A-129R) in the substrate-binding domain was replaced by the corresponding part of leucine dehydrogenase (M-D-I-I-Y-Q) in order to investigate the mechanism of substrate recognition by phenylalanine dehydrogenase. The catalytic efficiencies (kcat/Km) of the mutant enzyme with aliphatic amino acids and aliphatic keto acids as substrates were 0.5 to 2% of those of the wild-type enzyme. In contrast, the efficiencies for L-phenylalanine and phenylpyruvate decreased to 0.008 and 0.035% of those of the wild-type enzyme, respectively. These results suggest that the hexapeptide segment plays an important role in the substrate recognition by phenylalanine dehydrogenase.View full abstractDownload PDF (1235K) -
Hisanori Hara, Yoshinori Nakae, Toshiyuki Sogabe, Izumi Ihara, Syuji U ...1993 Volume 114 Issue 1 Pages 76-82
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structures of the N-linked oligosaccharides on recombinant human hepatocyte growth factor (rh-HGF) expressed by Chinese hamster ovary (CHO) cells were studied by two-dimensional sugar mapping. The oligosaccharides released from the glycopeptides by peptide: N-glycosidase F (PNGase F) treatment were tagged with 2-aminopyridine at the reducing ends. The α-chain was linked by biantennary, triantennary, and tetraantennary oligosaccharides, but the dominant oligosaccharides linking the β-chain were biantennary (>85%). There was no significant difference in oligosaccharide structures between the two glycosylation sites on each chain, that is, Asn263 and Asn371 on the α-chain, and Asn535 and Asn622 on the β-chain. The linkage of sialic acid to the non-reducing terminal galactose was identified as NeuAcα(2-3) by 1H-NMR spectrometry. The structures of the N-linked oligosaccharides from rat HGF were also studied. Triantennary oligosaccharides were obtained from the α-chain and a biantennary oligosaccharide was obtained from the β-chain. This result indicates that the α-chain is also linked by higher branched oligosac-charides than the β-chain in rat HGF.View full abstractDownload PDF (646K) -
Masahito Shimojo, Ryuzo Sakakibara, Masatsune Ishiguro1993 Volume 114 Issue 1 Pages 83-87
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman chorionic gonadotropin (hCG) in first trimester placental cells is made up of immature α- and β-subunits containing only N-linked high-mannose sugar chains, which are of 21 kDa for the α-subunit and 23 and 19 kDa for the β-subunit. However, the apparent molecular weight of immature hCG from placental cell extracts has been estimated from gel filtration to be much higher (100-200 kDa; high molecular weight-hCG, HMW-hCG) based on gel filtration than the theoretical value (-44 kDa) of the αβ dimer (αβ-hCG). We prepared a gel-filtered fraction containing HMW-hCG and investigated treatments for converting it to αβ-hCG. We found that the molecular weight of HMW-hCG was decreased to close to that of αβ-hCG by treatment with acetone, proteases, or chelating agents. These treatments also shifted the isoelectric point of HMW-hCG from the acidic region (pI=4-6) to the alkaline (pI=9-11), approximating to that of αβ-hCG. We also found that HMW-hCG, but not acetone-treated HMW-hCG, bound to ATP-agarose resin. These results suggested that the immature αβ-hCG molecule in placental cells may be bound to an acidic ATP-binding protein to form HMW-hCG.View full abstractDownload PDF (2248K) -
Isao Yumoto, Satoshi Takahashi, Teizo Kitagawa, Yoshihiro Fukumori, Ta ...1993 Volume 114 Issue 1 Pages 88-95
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome aco3, of Bacillus YN-2000 was purified by an improved procedure and its molecular features and catalytic activity were extensively studied. The enzyme molecule was composed of three subunits with Mrs of 50, 000, 41, 000, and 22, 000, and contains 1 molecule each of cytochrome α, cytochrome c, and cytochrome o3, in the minimal structural unit (Mr, 113, 000). The 41, 000 subunit (subunit II) contains heme c. The EPR, optical, and resonance Raman spectra of the oxidized enzyme demonstrated the presence of CuA, whose coordination environment bore close resemblance to that of the αα3-type cytochrome c oxidase. Resonance Raman studies demonstrated that the cytochrome a moiety was similar to that of an αα3-type oxidase and also that the cytochrome o3, contained a five-coordinated high-spin heme with histidine as an axial ligand. The Fe-CO stretching mode of the cytochrome o3•CO complex was observed at 520cm-1, which is the same frequency as that of cytochrome αα3•CO. The oxygen consumption activity of cytochrome aco3, was measured using several kinds of cytochromes c as the electron mediators. The reaction between cytochrome aco3 and eucaryotic cytochromes c was completely inhibited by poly-L-lysine. In contrast, poly-L-lysine was indispensable for sufficient reaction between the oxidase and Bacillus YN-2000 cytochrome c-553, the physiological electron donor. The combined results on the structure and enzymatic properties suggest that the cytochrome aco3, is very similar to cytochrome aco3 except that the cytochrome aco3 has cytochrome o3 in place of cytochrome α3 and the cytochrome c component has a very low redox midpoint potential (95 mV). A possible relationship between the role of the low redox potential cytochrome c and the bioenergetic properties of the alkalophilic bacterium is discussed.View full abstractDownload PDF (1920K) -
Kensaku Mizuno, Osamu Higuchi, Hisao Tajima, Tomoko Yonemasu, Toshikaz ...1993 Volume 114 Issue 1 Pages 96-102
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe proliferative potential of hepatocytes in the normal intact liver is highly suppressed, but they proliferate actively after liver injury. In this study, adult rat hepatocytes in primary culture were used to study mechanisms controlling hepatocyte growth in liver regeneration. DNA synthesis in hepatocytes cultured at a low cell density was highly stimulated in response to hepatocyte growth factor (HGF), but this stimulatory effect was not so obvious in hepatocytes cultured at a high cell density. In close parallel to the potency of DNA synthesis, the amounts of 125I-HGF specifically bound to hepatocytes cultured at a low cell density were much greater than in high cell density culture. Scatchard plots revealed that change in the specific binding of 125I-HGF was due to change in the number of high-affinity HGF receptors, but without change in the Kd, values. Affinity cross-linking of the HGF receptor with 125I-HGF confirmed the higher expression of HGF receptor on hepatocytes cultured at a lower cell density. Since there was no significant change in the expression of c-met mRNA in hepatocytes cultured at different cell densities, the number of cell surface HGF receptors is probably regulated by post-transcriptional mechanisms. We also found that the rates of HGF-induced down-regulation and recovery of HGF receptors on hepatocytes cultured at a low cell density were much faster than in cases of a high cell density. These results suggest that hepatocytes in the injured liver, where the cell-cell contact is loosened, as it is when cells are cultured at a low cell density, express a greater number of HGF receptors, which internalize in response to HGF, whereas hep-atocytes in the intact liver, where the cell-cell contact is tight, as it is for cells cultured at a high cell density, express a relatively low number of HGF receptors which have lost the ability to internalize and respond to HGF.View full abstractDownload PDF (1435K) -
Hiroshi Munakata, Takashi Yamagami, Tadashi Nagai, Masayuki Yamamoto, ...1993 Volume 114 Issue 1 Pages 103-111
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe existence of erythroid form δ-aminolevulinate synthase (ALAS-E) was historically a matter of some controversy. To obtain direct evidence for a unique ALAS-E, we have purified ALAS-E to homogeneity for the first time, from rat reticulocyte lysate. The papain digestion method was used at the initial step of the purification to overcome the difficulty which repeatedly hampered earlier attempts to purify ALAS-E. The size of the purified papain-resistant core catalytic domain of ALAS-E was estimated electrophoretically to be 49, 000 Da. The pH optimum (7.6) and apparent Km values for the substrates, glycine (6.5mM) and succinyl-CoA (2 μM), were similar to those of the non-specific form of δ-aminolevulinate synthase (ALAS-N); but, in contrast to ALAS-N, the substrate inhibition by succinyl-CoA was not evident in ALAS-E. We then isolated cDNA and genomic DNA clones encoding rat ALAS-E. By combining the nucleotide sequence information of the cDNA and genomic clones, the rat ALAS-E precursor is predicted to be composed of 587 amino acids with a calculated molecular mass of 64, 841 Da. All the peptide sequences determined directly from the purified protein agreed with those predicted from the nucleotide data, demonstrating the existence of ALAS-E. Analysis of the papain-resistant core domain further revealed that it overlaps with the evolutionally conserved segment that has been noticed by sequence alignment analysis of ALA synthases from various species.View full abstractDownload PDF (3085K) -
Hiroshi Sagami, Tatsumi Korenaga, Akira Kurisaki, Kyozo Ogura1993 Volume 114 Issue 1 Pages 112-117
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen assayed by the conventional method for prenyltransferase using a combination of [1-14C] isopentenyl and geranyl diphosphates, 100, 000×g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C] isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H] mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H] mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.View full abstractDownload PDF (1578K) -
Hiroshi Sagami, Tatsumi Korenaga, Kyozo Ogura1993 Volume 114 Issue 1 Pages 118-121
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGeranylgeranyl diphosphate synthase was purified 191-fold from bovine brain by Mono Q column chromatography followed by preparative isoelectric focusing electrophoresis and Superose 12 gel filtration. The synthase had a pI value at 6.0, and it was made free of farnesyl diphosphate synthase, the pI of which was 5.1. The partially purified enzyme catalyzed the formation of geranylgeranyl diphosphate from isopentenyl diphosphate and farnesyl diphosphate with the Km values for isopentenyl diphosphate and farnesyl diphos-phate being 14 and 0.8μM, respectively. Dimethylallyl diphosphate and geranyl diphos-phate were poor substrates with velocities of only 0.003 and 0.03, respectively, relative to that of farnesyl diphosphate. These results indicate that geranylgeranyl diphosphate synthase catalyzes a single condensation between isopentenyl diphosphate and farnesyl diphosphate and that farnesyl diphosphate is the common intermediate at the branch point for the synthesis of geranylgeranylated proteins as well as cholesterol, ubiquinone, dolichol, and farnesylated proteins. The enzyme required Mg2+ or Mn2+ for maximum activity. Octylglucoside showed a stimulatory effect on the enzyme activity.View full abstractDownload PDF (1149K) -
Yosuke Aoki, Tomomi Yamazaki-Hase1993 Volume 114 Issue 1 Pages 122-125
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElastinolytic activity of medullasin was investigated precisely and compared with that of human leukocyte elastase, because the structure of medullasin is quite similar to that of human neutrophil elastase, which was reported to have elastinolytic activity. When elastinolytic activity of medullasin and human leukocyte elastase was determined by employing unstained elastin fibers and measuring the increase in 280-nm absorbance of the supernatant, elastinolytic activity amounting to several percent of that of porcine pancreas elastase was apparently observed. However, the susceptibility of elastin preparations to these proteases was proportional to their hydroxyproline content. Both medullasin and human leukocyte elastase digested collagen fibers obtained from bovine Achilles tendon to the same extent as collagenase from Clostridium histolyticum. When elastinolytic activity was determined by employing elastin fibers stained with orcein, both proteases showed negligible elastinolytic activity. The activity remained negligible even when the pH or ionic strength of the reaction mixture was altered. These results indicate that medullasin and human leukocyte elastase are essentially devoid of elastinolytic activity, and that apparent elastinolytic activity observed when unstained elastin fibers were employed as the substrate is due to the digestion of collagen fibers mingled with elastin preparations.View full abstractDownload PDF (481K) -
Shun-ken Kim, Junji Ezaki, Masaru Himeno, Keitaro Kato1993 Volume 114 Issue 1 Pages 126-131
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatog-raphy, gel filtration with Superdex 200, DEAE ion exchange chromatography, and prepar-ative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140, 000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu-Ala-Leu-.View full abstractDownload PDF (2306K) -
Hideko Yamamoto, Yasuko Hagiwara, Yuji Mizuno, Mikiharu Yoshida, Eijir ...1993 Volume 114 Issue 1 Pages 132-139
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe proteins which compose the complex of dystrophin and its associated proteins were analyzed by two-dimensional PAGE, i.e., electrofocusing in the presence of 8M urea followed by SDS PAGE. Silver-staining of the gel showed many more spots than were expected from the results of one-dimensional SDS PAGE. By examination of their reactivity with specific antibodies, various lectins and 3-(trifluoromethyl)-3-(m-[125I] iodophenyl)-diazirine, most of these spots were identified as dystrophin and its associated proteins described previously. Several as yet unidentified minor proteins were also detected. Dystrophin-associated protein Al was separated into two groups, α-A 1 and β-A 1, both composed of numerous spots. These groups differed from each other in isoelectric point, molecular mass, and reactivity with antibodies. The β-A 1 group (64 kDa) was more basic than the α-A 1 group (60 kDa). Beta-A 1 but not α-A 1 reacted with several lectins, indicating that β-A 1 is a glycoprotein. This is incompatible with the report that 59DAG, which corresponds to A 1 (α-A 1+β-A 1), is not a glycoprotein [Ervasti et al. (1991) Cell 66, 1121-1131]. The charge heterogeneity observed in α-A 1 and β-A 1 may be partially due to differential phosphorylation. The charge heterogeneity of A2 and A3a may, at least to some extent, be due to differential sialilation of their carbohydrates.View full abstractDownload PDF (4420K) -
Masaki Iizuka, Kazuhiko Fukuda1993 Volume 114 Issue 1 Pages 140-147
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe bovine skeletal muscle nicotinic acetylcholine receptor α-subunit was produced in insect, Spodoptera frugiperda (Sf 9), cells by infection with a recombinant baculovirus. The expressed α-subunit protein could not be solubilized efficiently with Triton X-100 or sodium cholate, but could be solubilized efficiently with Zwittergent 3-14, sodium dodecyl sulfate or tris (hydroxymethyl) aminomethane dodecylsulfate. After solubilization of the α-subunit protein with Zwittergent 3-14 from the Triton X-100-insoluble fraction, the α-subunit protein was purified by concanavalin A-Sepharose chromatography and DEAE ion-exchange chromatography. A milligram quantity of the α-subunit protein could be purified from 8g (wet weight) of Sf 9 cells infected with the recombinant baculovirus. Chromatographic analyses including hydroxyapatite chromatography, DEAE ion-exchange chromatography, gel filtration chromatography and chromatofocusing and sucrose density gradient centrifugation analysis suggest that the purified α-subunit protein is homogeneous. The purified α-subunit protein had a high affinity for 125I-α-bungarotoxin and was glycosylated with a high mannose-type N-linked oligosaccharide side chain. These results indicate that purification of ion channel proteins produced by the baculovirus expression system is a promising approach to structural analysis of ion channel proteins, which are extremely rare membrane proteins in native tissues.View full abstractDownload PDF (2652K) -
Junji Nishioka, Hiroshi Taneda, Koji Suzuki1993 Volume 114 Issue 1 Pages 148-155
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Thrombin has several characteristic module structures around its active center. Previ-ously we showed that a synthetic peptide, TWTANVGKGQPS, corresponding to the residues Thr147 to Ser158 of the B-chain of human thrombin, a possible interaction site of thrombomodulin near the active center of thrombin, specifically blocked the interactions between thrombin and thrombomodulin, fibrinogen, Factor V, or platelets [Suzuki, K. & Nishioka, J. (1991) J. Biol. Chem. 266, 18498-18501]. To elucidate further the role of the other module structures, we studied the effects of several synthetic peptides; FRKSPQELL, LLYPPWDKNF, RIGKHSRTRYER, LEKIYIHP, RYNWREN, DSTRIRI, EGDSGGP, and SWGEGCDRDGK, respectively corresponding to the residues Phe19 to Leu27, Leu45 to Phe54, Arg62 to Arg73, Leu81 to Pro88, Arg89 to Asn95, Asp175 to Ile181, Glu202 to Pro208, and Ser226 to Lys236 of the B-chain of human thrombin, which are located around the active center, as well as TWTANVGKGQPS, on the interaction between thrombin and thrombomodulin, protein C, fibrinogen, Factor V, antithrombin III, or hirudin. Thrombin-thrombomodulin interaction was inhibited significantly by RYNWREN as well as TWTANVGKGQPS, and partially by LLYPPWDKNF. The inhibitory effects of the two former peptides were additive and thrombomodulin directly bound to them. RYNWREN and TWTANVGKGQPS also in-creased the Km values 3-7 times for protein C as compared with the conditions without peptide. Thrombin-induced protein C activation in the absence of thrombomodulin was specifically blocked by EGDSGGP. Thrombin-induced fibrinogen clotting was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS at lower concentrations, and by RYNWREN and DSTRIRI at higher concentrations. Thrombin-induced Factor V activation was blocked by FRKSPQELL, RIGKHSRTRYER as well as TWTANVGKGQPS. Thrombin inhibition by antithrombin III was blocked by RIGKHSRTRYER and RYNWREN. Thrombin inhibition by hirudin was blocked by FRKSPQELL, RIGKH-SRTRYER, and TWTANVGKGQPS. These findings suggest that the module structures around the active center of thrombin serve as recognition sites for several protein substrates playing a role in procoagulation or anticoagulation, and may restrict substrate specificity.View full abstractDownload PDF (1289K) -
Kumi Hayano, Masanori Iwama, Hiroko Sakamoto, Hideaki Watanabe, Akihir ...1993 Volume 114 Issue 1 Pages 156-162
Published: July 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly C preferential RNase previously reported by Levy and Karpetsky [J. Biol. Chem. 255, 2153-2159 (1980)] and Miura et al. [Chem. Pharm. Bull. 32, 4053-4060 (1984)] was extensively purified from chicken liver to homogeneity as determined by SDS-PAGE (RNase CL2). The poly C preference over poly U was slightly higher than that of bovine pancreatic RNase A. However, the kinetic constants for 8 dinucleoside phosphates, CpY and UpY (Y=one of A, G, U, and C) as substrates showed that RNase CL2 was preferential for cytidylic acid, but less so than RNase A, and the influence of Y base on the rate of hydrolysis of CpY or UpY was less marked than in the case of RNase A. The primary structure of RNase CL2 was determined. The molecular weight calculated from the sequence was 13, 420. Comparison of the amino acid sequence of RNase CL2 with those of other vertebrate RNases showed that RNase CL2 is a member of the RNase A family, but is not a non-secretory RNase. It retains 3 disulfide bridges of RNase A, but Cys65-Cys72 of RNase A is missing. As for the active site, the amino acid residues of the P0 and P1 sites of RNase A are completely conserved. Among the B1 site components, Thr45 (RNase A numbering) is conserved, but Phe120 and Ser123 are substituted by Leu and Thr, respectively. Among the B2 site residues, Gln69, Asn71 and Glu111 are substituted by other amino acids.View full abstractDownload PDF (697K)
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