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Volume 110 (1991)
- Issue 6 Pages 847-
- Issue 5 Pages 677-
- Issue 4 Pages 469-
- Issue 3 Pages 315-
- Issue 2 Pages 163-
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Volume 110, Issue 5
Displaying 1-30 of 30 articles from this issue
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Takeshi Itoh, Ken-ichi Tomita, Toshio Hakoshima, Hidekazu Hiroaki, Sei ...1991 Volume 110 Issue 5 Pages 677-680
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 Å resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHz NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].View full abstractDownload PDF (368K) -
Toshiyuki Mikami, Nobuo Tanaka, Takao Sato, Hideaki Moriyama, Masahiko ...1991 Volume 110 Issue 5 Pages 681-682
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHydroxylamine oxidoreductase [EC 1. 7. 3. 4] from Nitrosomonas europaea was crystallized by the microdialysis method using ammonium sulfate. Its space group is P63 with cell dimensions of a=b=96.4 Å and c=266.2 Å. Its molecular weight was determined to be 190, 000-195, 000 by the X-ray small angle scattering and ultracentrifugal methods.View full abstractDownload PDF (403K) -
Ken Karasawa, Noriko Satoh, Megumi Masuda, Morio Setaka, Kikuo Hashimo ...1991 Volume 110 Issue 5 Pages 683-687
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentaclecy1)-2-O-acetyl-sn-glycero-3-phosphocholine (ace-tyl-CPGPC), 1-O-(15'-carboxypentadecy1)-2-O- N, N-dimethylcarbamoyl- sn-glycero-3- phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O-(15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-phosphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl>> dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethyl-carbamoyl>acetyl>butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC-BSA; and butylcarbamoyl>dimethylcarbamoyl>acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lyso-phosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these anti-bodies. Anti-dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG in-hibited a PAF-induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG.View full abstractDownload PDF (937K) -
Yasutake Shimizu, Hideki Nikami, Masayuki Saito1991 Volume 110 Issue 5 Pages 688-692
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of norepinephrine (NE) infusion and surgical denervation or electrical stimula-tion of the sympathetic nerves on 2-deoxyglucose (2-DG) uptake in interscapular brown adipose tissue (BAT) were investigated in vivo in rats to obtain direct evidence for sympathetic control of glucose utilization in this tissue. 2-DG uptake was rather low in fasted rats, but after refeeding it increased in the BAT as well as the heart, skeletal muscle, and white adipose tissue, in parallel with an increase in plasma insulin level. Cold exposure also enhanced 2-DG uptake in the BAT without the increase in plasma insulin level, while it had no appreciable effect on 2-DG uptake in other tissues. Sympathetic denervation greatly attenuated the stimulatory effect of cold exposure on 2-DG uptake in BAT, but it did not affect the increased 2-DG uptake after refeeding. Electrical stimulation of the sympathetic nerves entering BAT or NE infusion produced a marked increase in 2-DG uptake in BAT without noticeable effects in other tissues. β-Adrenergic blockade, but not α-blockade, abolished the increased 2-DG uptake in BAT. It was concluded that glucose utilization in BAT is activated directly, independently of the action of insulin, by sympa-thetic nerves via the β-adrenergic pathway.View full abstractDownload PDF (430K) -
Shiho Kodama, Tamao Endo, Nobuo Tsuruoka, Masafumi Tsujimoto, Akira Ko ...1991 Volume 110 Issue 5 Pages 693-701
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe asparagine-linked sugar chains of recombinant human interleukin 5 produced by Chinese hamster ovary cells were released quantitatively as oligosaccharides by hydra-zinolysis. After N-acetylation followed by NaB3H4 reduction, each oligosaccharide was isolated by paper electrophoresis and serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion in combination with methylation analysis, revealed that they are bi-, tri-, and tetraantennary complex-type with fucosylated and non-fucosylated trimannosyl cores and high mannose type sugar chains. More than 8O% of the sugar chains occur as biantennary complex-type sugar chains. Although acidic oligosaccharides amount to only 14% of the total oligosaccharides, their sialic acid residues occur exclusively as the Siaα2→3Gal group. Removal of the sugar moiety from intact recombinant human interleukin 5 produced a 2.5-fold increase of its activity to induce IgM secretion.View full abstractDownload PDF (1284K) -
Francoise Gasnier, Hélène Baubichon-Cortay, Pierre Louis ...1991 Volume 110 Issue 5 Pages 702-707
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrevious studies have shown that purified mitochondrial outer membrane is able to catalyze the transfer of sialic acid from CMP-Neu5Ac to an exogenous asialoglycoprotein acceptor, asialofetuin. Considering the heterogeneity of the glycan chains borne by this glycoprotein, an investigation of mitochondrial sialyltransferase activities was under-taken. Our data provide evidence for the existence of two distinct sialyltransferases in purified mitochondrial outer membranes. The use of different acceptor substrates, the temperature dependence of these enzymes, and their different sensitivity towards a sulfhydryl reagent, p-CMB, allowed us to discriminate between a galactoside α(2-3) sialyltransferase and a galactoside α(2-6) sialyltransferase presumably involved in the sialylation of O- and N-glycan chains of glycoprotein, respectively. These results are discussed in terms of mitochondrial autonomy for post-translational events.View full abstractDownload PDF (1060K) -
Yasuaki Kazuta, Katsuyuki Tanizawa, Toshio Fukui1991 Volume 110 Issue 5 Pages 708-713
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy using two reactive analogues of UDP-Glc, uridine di- and triphosphopyridoxals, we have recently probed the substrate-binding site in potato tuber UDP-Glc pyrophosphorylase [EC 2. 7. 7. 9]. In this work, pyridoxal diphospho-α-D-glucose was used for the same purpose. This compound is also a reactive UDP-Glc analogue but having its reactive group on the opposite side of the pyrophosphate linkage to those of the above two compounds. The enzyme was rapidly inactivated when incubated with the compound at very low concentrations followed by reduction with sodium borohydride. The inactivation was almost completely prevented by UDP-Glc and UTP. Complete inactivation corresponded to the incorporation of 1. 0 mol of the reagent per mol of enzyme monomer. The label was found to be distributed in five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410). All of these results were similar to those obtained previously with the other compounds, suggesting the presence of a cluster of five lysyl residues at or near the substrate-binding site of this enzyme. However, the incorporations of labels into each lysyl residue differed depending on the compounds used. The substrate retarded the incorporations in different manners. Based on the combined results of the present and previous studies, a hypothetical model is presented for the possible locations of the five lysyl residues around the substrate bound to the enzyme. This model is consistent with the kinetic properties of mutant enzymes in which the five lysyl residues were individually replaced by glutamine via site-directed mutagenesis.View full abstractDownload PDF (580K) -
Akiyoshi Uchiyama, Tsuyosi Ohishi, Masaaki Takahashi, Kazuhiro Kushida ...1991 Volume 110 Issue 5 Pages 714-718
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman articular cartilages of various ages were digested with collagenase, and the fluorescence of the digests was measured as a function of age. At acidic pH, all collagenase-treated fractions were found to contain two main fluorophores with fluorescence maxima at 395 and 385 nm (excitation at 295 and 335 nm, respectively). Each fluorophore was isolated from the hydrolysate and its structure was deduced from spectral and chemical data. The 395/295 nm fluorophore was identified as pyridinoline, which is one of the non-reducible cross-linkages in collagen. The 385/335 nm fluorophore was identical to pentosidine, which was isolated from human dura mater and characterized by Sell and Monnier in 1989. Our results showed that the amount of pentosidine per collagen in human articular cartilage increases linearly with age (r=0.929, p<0.005), while the amount of pyridinoline per collagen remained constant and was not correlated with age (r=0.20). On the other hand, the amount of pentosidine per pyridinoline increased exponentially during life (r2=0.839, p<0.05).View full abstractDownload PDF (435K) -
Tadashi Ueda, Hidenori Yamada, Noriko Sakamoto, Yoshito Abe, Keiichi K ...1991 Volume 110 Issue 5 Pages 719-725
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the β-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1, 3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a-CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3°C than that of native lysozyme at pH 3.View full abstractDownload PDF (672K) -
Takumi Hatayama, Yasuhiro Tsukimi, Tohru Wakatsuki, Teruko Kitamura, H ...1991 Volume 110 Issue 5 Pages 726-731
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo clarify the physiological roles of heat shock proteins induced by copper, we studied the synthesis of these proteins and metallothionein, as well as the level and nature of copper incorporated into HeLa cells. Incubation in medium containing 200 μM cupric sulfate and above induced the synthesis of 70, 000-Da heat shock protein (hsp70) in these cells. However, the synthesis of hsp70 did not increase in the presence of less than 200 μM cupric sulfate. On the other hand, the synthesis of metallothionein increased due to 100 μM cupric sulfate. The uptake of copper into the cells depended on the cupric sulfate concentration in the medium. To analyze the nature of the intracellular copper, cell extracts were separated by gel filtration chromatography into three fractions: the high molecular weight, metallothionein, and low molecular weight fractions. No copper was found in the low molecular weight fraction of control cells, but appeared distinctly at 200 μM cupric sulfate and above. Copper in the high molecular weight fraction also began to increase at 200 μM cupric sulfate and above, whereas in the metallothionein fraction it began to increase even at 50 to 100 μM cupric sulfate. Furthermore, inhibition of cell growth was also observed at 200 μM cupric sulfate and above but not at 100 μM and below. Since the induction of hsp70 synthesis and inhibition of cell growth occurred concomitantly with the increase of copper in the high and low molecular weight fractions, hsp70 seemed not to participate in the detoxification of copper as does metallothionein, although it may function in repairing damage caused by the metal.View full abstractDownload PDF (1423K) -
Takako Nishiya, Hui Li Chou1991 Volume 110 Issue 5 Pages 732-736
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of melittin on the bilayer-to-inverted hexagonal (H11) phase transition of egg phosphatidylethanolamine (EPE) and the influence of the phase state of membrane matrix on hydrolysis of EPE by phospholipases have been studied. The phase transitions were measured using the fluorescent probe N-(7-nitro-2, 1, 3-benzoxadiazol-4-yl)phosphatidyl-ethanolamine (N-NBD-PE) and differential scanning calorimetry. In the presence of melittin at a lipid-to-melittin molar ratio (R1) of 200, 100, and 20, the phase transition of EPE disappeared, indicating that melittin stabilizes the bilayer structure. In the presence of 10 mol% of cholesterol, the phase transition temperature (TH) decreased and TH was observed even in the presence of melittin at R1 of 200 and 100. The fluorescence intensity of the tryptophan residue of melittin is sensitive to the phase transition and the wavelength of emission maxima shift from 352 to 337 nm upon addition of EPE and EPE-cholesterol (10 mol%) at R1 of 200. Kinetic parameters for phospholipase-catalyzed hydrolysis of EPE in bilayer and H11 phases showed that H11 phase of EPE is a poorer substrate for phospholipases and that cholesterol decreases the susceptibility of EPE to phospholipases.View full abstractDownload PDF (454K) -
Shin-ya Ohki, Michio Yazawa, Koichi Yagi, Kunio Hikichi1991 Volume 110 Issue 5 Pages 737-742
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction between calmodulin and mastoparan at various concentrations of calcium ions was studied by 1H NMR. It was found that at lower mastoparan concentrations 1mol of mastoparan binds to both the C-terminal-half and N-terminal-half regions of calciumsaturated calmodulin. The mastoparan affinity is much greater for the C-terminal-half region than for the N-terminal-half region. At higher mastoparan concentrations, a further 1 mol of mastoparan binds to the N-terminal-region of calcium saturated calmodulin. The results can be interpreted in terms of the assumption that the N-terminal-half region of calmodulin with mastoparan has a higher calcium ion affinity than the C-terminal-half region without mastoparan. It is suggested that calcium ions transfer from the C-terminal-half region of calmodulin without mastoparan to the N-terminal-half region of calmodulin with mastoparan. This calcium ion transfer is discussed from the viewpoint of enzyme activation by calmodulin.View full abstractDownload PDF (542K) -
Makaki Kashiwada, Mitsukazu Kitada, Tomomi Shimada, Koshiro Itahashi, ...1991 Volume 110 Issue 5 Pages 743-747
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4. 8 and 4. 9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23, 000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2, 4-dinitro-benzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-π. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-π from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.View full abstractDownload PDF (1492K) -
Akiko Kita, Nobutami Kasai, Sabu Kasai, Tadao Nakaya, Kunio Miki1991 Volume 110 Issue 5 Pages 748-750
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA flavoprotein, FP390, obtained from a luminescent bacterium, Photobacterium phosphoreum, in the purification of luciferase has been crystallized by the vapor-diffusion procedure. Crystals obtained from polyethylene glycol 4000 solutions, whose X-ray photographs show powder diffraction patterns, were unsuitable for further crystallo-graphic work. However, tetragonal crystals grown from potassium phosphate solution well diffracted X-rays beyond 3Å resolution. The space group of this crystal is P4122 or P4322 with unit-cell dimensions of a=b=76.8 and c=241Å. Assuming two or three molecules in an asymmetric unit, the value for the crystal volume per unit molecular mass, Vm, is calculated as 3.3 or 2.2Å3/Da, respectively. A total of 13, 555 independent reflections for the native crystal was collected up to 3Å resolution using a Weissenberg camera attached to the synchrotron radiation source, the merging R factor being 0.077 for 79, 335 measurements.View full abstractDownload PDF (1225K) -
Mikiko Yasuhara, Tomoko Ohama, Norio Matsuki, Hiroshi Saito, Teruhiko ...1991 Volume 110 Issue 5 Pages 751-755
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe reported previously that fatty liver is easily induced in a novel experimental animal, Suncus murinus (suncus) by withholding food. In this study, we focused on lipoprotein and apolipoprotein secretion from the liver. The study of lipoproteins from this animal revealed that small amounts of lipoproteins with apolipoprotein (apo) E but without apo B were observed in the fraction of density less than 1.08g/ml. In order to learn whether apo B is synthesized by the liver or not, isolated suncus livers were perfused with an addition of [35S]methionine. Small amounts of radioactivity were observed in apo E of VLDL, and fairly large amounts in apo E and A-I in the fraction of LDL+HDL, suggesting that VLDL was secreted with apo E but not with apo B from the liver. Northern blot analysis with use of rat apo B cDNA revealed a weak signal of hybridized rat apo B cDNA between 15kb and 9kb in the suncus liver and intestinal mucosa; this is almost the same size as rat apo B mRNA. This finding suggests the presence of apo B mRNA in the suncus. In conclusion, apo B is not secreted from the suncus liver, owing to a defect in intracellular post-transcriptional processing or to ineffective transcription. This might be one of the reasons for fatty deposits in the suncus liver. Suncus may be a candidate for an animal model of abetalipo-proteinemia as well as fatty liver due to a defect of apo B synthesis.View full abstractDownload PDF (1552K) -
Sou Abe, Yuko Nagamine, Kaoru Omichi, Tokuji Ikenaka1991 Volume 110 Issue 5 Pages 756-761
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active site of Bacillus macerans cyclodextrin glucanotransferase (CGTase) was examined by use of derivatives of phenyl α-maltopentaoside and phenyl α-glucoside as the substrates and acceptors, respectively. The active site of this enzyme is considered to be composed of tandem subsites (S4, S3, S2, S1, S1', S2', etc.) geometrically complementary to several glucose residues, and the α-1, 4-glycosidic linkage of a substrate is split between S1 and S1'. The features of subsites S3 and S4 of the glycon binding site were estimated from the modes of the enzymatic action on phenyl α-maltopentaoside (G-G-G-G-G-Φ; G, glucose residue; Φ, phenyl residue; -, α-1, 4-glycosidic bond) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2I (IG-G-G-G-G-Φ; IG, 6-deoxy-6-iodo-D-glucose residue), CH2NH2 (AG-G-G-G-G-Φ; AG, 6-amino-6- deoxy-D-glucose residue), or COOH (CG-G-G-G-G-Φ; CG, glucuronic acid residue). p-Nitro-phenyl α-glucopyranoside (G-P; P, p-nitrophenyl residue) was used as an acceptor. HPLC analysis of the digests revealed that the CG residue of CG-G-G-G-G-Φ was excluded from subsite S3, while it was accommodated in subsite S4. The Km and Vmax, values for CG-G-G-G-G-Φ were remarkably larger and smaller, respectively, than those for any other substrates. The results suggested that the CH2R groups (R:OH, I, NH2) have interactions with subsite S3 and/or the COOH group interupts binding of the CG residue to subsite S3. The features of subsite S1' were examined using phenyl α-D-glucopyranoside derivatives [G-Φ, IG-Φ, DG-Φ (DG: 6-deoxy-D-glucose residue), AG-Φ, or CG-Φ] as acceptors. FG4P (FG-G-G-G-P, FG: 6-deoxy-6-[(2-pyridy)Damino]-D-glucose residue) was used as the substrate. The product analysis showed that the extent of transfer reaction to produce FG-G-G-XG-Φ decreased in the order of G-Φ, IG-Φ, DG-Φ, AG-Φ, and CG-Φ, and the extent of hydrolysis to give FG-G-G and GP increased in the same order. These data suggested that subsite S1' excluded the charged groups, especially negatively charged groups, of modified acceptors, and CGTase catalyzed mainly the transfer reaction in the presence of a suitable acceptor, and the hydrolysis reaction with unsuitable acceptors.View full abstractDownload PDF (513K) -
Michiaki Yamashita, Shiro Konagaya1991 Volume 110 Issue 5 Pages 762-766
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo different cysteine protease inhibitors (Forms I and II) were isolated from chum salmon egg, and their molecular weights were found to be 16, 000 and 11, 000, respectively. When the N-terminal amino acid sequences of Forms I and II were compared, 67% of the residues were identical but no apparent homology was found between these inhibitors and cystatins of higher vertebrates. Because the amounts of half-cystine were estimated to be 7-8 residues per molecule, they can be classified into a new group of the cystatin superfamily.View full abstractDownload PDF (780K) -
Yoshio Yamakawa, Tamotsu Omori-Satoh1991 Volume 110 Issue 5 Pages 767-769
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new method is presented for purifying antihemorrhagic factor in the serum of Japanese Habu snake (Trimeresurus flavoviridis). The method consists of specific binding of the factor to a hemorrhagic principle-conjugated Sepharose, gel filtration and reverse-phase HPLC. The improved method is simple and rapid, providing the factor with a great increase in specific activity and in a high yield.View full abstractDownload PDF (551K) -
Teruo Miyauchi, Yasushi Masuzawa, Takashi Muramatsu1991 Volume 110 Issue 5 Pages 770-774
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBasigin belongs to the immunoglobulin superfamily and may be related to the primordial form of the superfamily. Human basigin cDNA was isolated and sequenced, and the predicted protein structure was compared with that of mouse basigin and two related molecules, embigin and the chicken blood-brain barrier antigen HT7. Between human and mouse basigin, 58% of the amino acids were identical and 80% of the changes were conservative. A stretch of 29 amino acid residues in the transmembrane and cytoplasmic domains was conserved not only in human and mouse basigin but in HT7 antigen. The conserved structure may be required for interaction with a membranous protein. In addition, the relationship of basigin with other members of the immunoglobulin superfamily has been evaluated.View full abstractDownload PDF (719K) -
Tsukasa Fueki, Kazuo Yamaguchi1991 Volume 110 Issue 5 Pages 775-779
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurified replication initiator protein (Rep) of plasmid pSC101 binds preferentially to two inverted repeats (IR) overlapping the promoter of its own structure gene, rep. However, the protein has much lower binding affinity for directly repeated (DR) sequences in the replication origin (ori) that are similar to the symmetric sequences. Exonuclease III (exo III) promotes in vitro binding of Rep to the origin repeats. In the present studies, DNA containing the DR sequences was degraded unidirectionally by exo III and then formed a complex with Rep. Analyses of DNA from the complex revealed that Rep bound to the DR sequences only when the degradation proceeded from the 3' end proximal to IR to the DR sequences, resulting in conversion of the duplex structure in a specific downstream region of DR into the single-stranded form. The degradation in the opposite direction had no effect on binding of Rep. These results suggest that a localized structural change of DNA adjacent to DR is required for Rep binding to double-stranded DR sequences. By contrast, exo III strikingly inhibited binding of Rep to DNA containing the IR sequences by introducing a single-stranded moiety into duplex IR sequences.View full abstractDownload PDF (1243K) -
Eiichi Tanaka, Kinuko Kimura1991 Volume 110 Issue 5 Pages 780-784
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfo-nylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8- N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn2+-ATP and that binding Mg2+-ATP was detected by affinity labeling with ATP analogs.View full abstractDownload PDF (435K) -
Haruki Yamaguchi1991 Volume 110 Issue 5 Pages 785-789
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn α-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The α-subunit was free from tryptophan and cysteine and the β-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The α-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosac-charides, whereas the sugar moiety of the β-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of α- and β-subunits were shown to be 7, 800 and 14, 000, respectively. These values were consistent with molecular sizes obtained for α- and β-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28, 800, obtained by gel permea-tion HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.View full abstractDownload PDF (807K) -
Yusuke Mizuno, Yoshikazu Kanesaka, Hisakazu Fujita, Osamu Minowa, Hiro ...1991 Volume 110 Issue 5 Pages 790-794
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElectrophoretically homogeneous preparations of organ-common type acylphosphatase from porcine testis and brain were separated into two molecular species by reversed-phase liquid chromatography. From tryptic peptide map analysis, it was inferred that each of the two testis proteins is the same as the corresponding one of the two brain proteins. The complete primary structures of the two acylphosphatases from testis were then determined. The one molecular species consists of 100 amino acid residues:N-acetyl-SMAEGDTLISVD-YEVEGKVQGVFFRKYTQAEGKKLGLVGWVQNTDQGTVQGQLQGPTSKVIIHMQEWLET-RGSPKSHIDRASENNEKVISKLDYSDFQIVK. The other consists of 98 amino acid residues identical to the 3rd-100th residues of the above sequence and is also acetylated at the amino-terminal alanine. The 98-residue sequence has only 59% homology with porcine muscle acylphosphatase, but has 92% homology with human erythrocyte acylphosphatase. It was thus confirmed that the major acylphosphatases in testis, brain, and erythrocyte belong to the same organ-common type isoenzyme, distinct from the muscle type isoen-zyme.View full abstractDownload PDF (439K) -
Kazuo Inaba, Kazuo Ogawa, Hideo Mohri1991 Volume 110 Issue 5 Pages 795-801
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe β chain of sea urchin outer-arm dynein showed a peculiar tryptic digestion pattern in the presence of ATP (or ADP) plus V1. Examination of the molecular mass of the products formed by photocleavage of tryptic fragments indicated that the trypsin-sensitive sites on the 165-kDa ATP-binding polypeptide in the presence of ATP and V1 are located 15 kDa apart from its amino-terminus, 2 kDa apart from its carboxy-terminus, and near the middle portion between the adenine- and γ-P1-binding sites. On the other hand, the carboxy-terminal region of the β chain, the 135-kDa polypeptide, was cleaved into a 96-kDa polypeptide by tryptic digestion in the presence of ATP and V1. Peptide mapping of 135-kDa, 96-kDa, and carboxy-terminally truncated polypeptides of the 135-kDa polypep-tide revealed that the 96-kDa region is located at the amino-terminal portion of the 135- kDa region. These results indicate that the changes of trypsin susceptibility of dynein β chain caused by binding ADP and V1 occur not in local region but over an extensive region on the β chain.View full abstractDownload PDF (1802K) -
Sung-Woo Cho, Geum-Yi Kim1991 Volume 110 Issue 5 Pages 802-805
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid ribonuclease (optimum pH 6.0) has been purified from bovine brain in a five-step procedure. The preparation appeared homogeneous on SDS-polyacrylamide gel electro-phoresis. The molecular size of the acid ribonuclease is 70 kDa and it is a dimeric protein with a subunit molecular size of 35 kDa. The acid RNase was activated by aluminum at low concentration. Preincubation of the acid RNase with 10μM aluminum increased the specific activity of the enzyme 2.3-fold at acid pH, while the effect of aluminum was much weaker at alkaline pH under otherwise the same conditions. A stoichiometry of 1 : 1 for the binding aluminum to brain acid RNase was estimated. None of the enzyme-bound aluminum was dissociated by dialysis against 50 mM HEPES, pH 7.0 at 4°C for 24 h. Citrate, EDTA, NaF, and apotransferrin abolished the effects of aluminum on the enzyme. Ribonucleic acid also protected the enzyme against the activation caused by aluminum. These results suggest that accumulation of aluminum in brain may change the regulation of ribonucleic acid metabolism.View full abstractDownload PDF (865K) -
Masami Nagahama, Jo Ikemizu, Yoshio Misumi, Yukio Ikehara, Kazuo Murak ...1991 Volume 110 Issue 5 Pages 806-811
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is well known that precursor cleavage at paired basic amino acids (e.g., Lys-Arg, Arg-Arg) within the regulated secretory pathway is one of the key steps to produce bioactive peptides. On the other hand, we have recently shown that precursors with an Arg residue at the fourth residue upstream of the cleavage site besides the basic pair, i.e. with the Arg-X-Lys/Arg-Arg (RXK/RR) motif, are cleaved within the constitutive secretory pathway. To discriminate between the precursor cleavage at RXK/RR sites within the constitutive pathway and that at dibasic sites within the regulated pathway, we examined the effects of drugs affecting the secretory process, intracellular Ca2+ depletion, and a protease inhibitor on these cleavages. Chloroquine (a weak base), depletion of intracellular Ca2+ by A23187 (a Ca2+ ionophore), and the Pittsburgh-type mutant of α1-protease inhibitor differentially affected these two cleavages. Brefeldin A, which impedes protein transport from the endoplasmic reticulum to the Golgi complex, inhibited both cleavages. Colchicine (an anti-microtubular drug) had no discernible effect on either cleavage. These observations support the notion that the precursor cleavages at dibasic and RXK/RR sites occur in different subcellular compartments, and are catalyzed by different processing endoproteases.View full abstractDownload PDF (1212K) -
Yoriko Atomi, Shigeru Yamada, Richard Strohman, Yoshiaki Nonomura1991 Volume 110 Issue 5 Pages 812-822
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAtrophy of rat soleus muscles by hindlimb suspension is characterized by an early dramatic decrease in a soluble 22-kDa protein. The 22-kDa protein was purified from rat red skeletal muscle and rat lens by three different methods of chromatography. The partial amino acid sequence (65% of total amino acids) determined for muscle 22-kDa protein was identical with that of rat lens crystallin. The HPLC elution patterns of lysylendopeptidase fragments of 22-kDa protein from the two sources were identical. Polyclonal antibodies to rat muscle and bovine lens αB-crystallin with the two proteins on immunoblotting. αB-Crystallin protein was expressed and synthesized efficiently in slow skeletal muscle and poorly in fast muscle. Thus, the decreased 22-kDa protein of slow muscle in the suspension treatment was confirmed to be αB-crystallin. Immunoblotting confirmed that most of the αB-crystallin was solubilized, though some was tightly bound to myofibrils. This bound portion was localized in Z-bands of isolated myofibrils by immunocytochemical light and electron microscopy. Muscle αB-crystallin is tentatively proposed to be a myofibril-stabilizing protein, based upon its extraction characteristics, localization, and amino acid sequence.View full abstractDownload PDF (2675K) -
Shigeru Tsuboi, Tatsuko Suga, Kunio Takishima, Gunji Mamiya, Kazuhiko ...1991 Volume 110 Issue 5 Pages 823-831
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour kinds of nonspecific lipid transfer proteins (nsLTP) were purified from different organs of castor bean (Ricinus communis L. ) seedlings. Amino acid compositions and amino-terminal sequences of the four nsLTPs were determined and compared with those of castor bean isoforms, nsLTP-A, -B, and -C, previously reported [Takishima et al. (1986) Biochim. Biophys. Acta 870, 248-255; Takishima et al. (1988) Eur. J. Biochem. 177, 241-249]. Two isoforms from the cotyledons were identified as nsLTP-A and -C, one isoform from the endosperms as nsLTP-B, and the other was a new isoform from the axes. This new isoform was named nsLTP-D and its amino acid sequence was determined. These results demonstrated organ-specific occurrence of the nsLTP isoforms in castor bean seedlings. The isoforms nsLTP-A, -B, -C, and -D showed similar transfer activity not only for phospha-tidylcholine and phosphatidylethanolamine but also for monogalactosyldiacylglycerol, although the homology among their amino acid sequences ranged from 70 to 30%. Two cDNA clones (pnsLTP-C and pnsLTP-D) for nsLTPs of castor bean seedlings were isolated and sequenced. pnsLTP-C was the cDNA clone for nsLTP-C expressed in the cotyledons, and pnsLTP-D was that for nsLTP-D in the axis. A coupled in vitro transcription-translation analysis of both cDNA clones revealed that pnsLTP-C encodes the full-length of nsLTP-C precursor (pro-nsLTP-C), while pnsLTP-D encodes a part of nsLTP-D precursor. Pro-nsLTP-C contained a 24-amino acid pre-sequence preceding the mature nsLTP-C (92 amino acids). Northern blot analyses of mRNAs from castor bean organs for nsLTP-C and -D demonstrated the organ-specific expression of nsLTPs; nsLTP-C was mainly expressed in the cotyledons and nsLTP-D in the axis, although small amounts of nsLTP-C and nsLTP-D were expressed in the axis and cotyledons, respectively.View full abstractDownload PDF (2219K) -
Kyoko Nakamura, Minoru Suzuki, Choji Taya, Fuyuhiko Inagaki, Tamio Yam ...1991 Volume 110 Issue 5 Pages 832-841
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe disialogangliosides of WHT/Ht mouse thymomas, which were obtained by subcutane-ous transplantation of a thymoma that developed spontaneously in a WHT/Ht mouse, were purified and characterized. From the results of sugar-composition analysis, a permethyla-tion study, enzymatic hydrolysis followed by TLC-immunostaining, negative-ion fast atom bombardment mass spectrometry (FAB/MS), and 1H-NMR spectroscopy, the structure of one of the five purified disialogangliosides was determined to be IV3α(NeuGcα2-8NeuGc)-Gg4Cer. The other 4 disialogangliosides were tentatively characterized on the basis of sialidase treatment followed by TLC-immunostaining with cholera toxin B subunit and anti-Gg4Cer antibody to be IV α(NeuAc α-NeuGc)-Gg4Cer, IV α(NeuGc α-NeuAc)-Gg4Cer, IVαNeuAc, II3 αNeuAc-Gg4Cer, and IVαNeuGc, II3αNeuGc-Gg4Cer. In addition, another component exhibiting one spot on TLC was a mixture of IVαNeuGc, II3 αaNeuAc-Gg4Cer and IVαNeuAc, II3αNeuGc-Gg4Cer. Then the occurrence of these gangliosides in WHT/Ht mouse thymocytes was examined. As one of two major disialogangliosides, the thymocytes contained IV3 α(NeuGcα2-8NeuGc)-Gg4Cer, which was characterized with a mass spectrum and mass chromatograms obtained by micro high-performance liquid chromatography-FAB / MS. The other major disialoganglioside was tentatively characterized to be II3 α - (NeuGcα-NeuGc)-Gg4Cer by sialidase treatment followed by TLC-immunostaining. A sialidase-susceptible monosialoganglioside, IV3 αNeuGc-Gg4Cer [GM1b(NeuGc)], had been reported to be characteristic of mouse immune tissues [Nakamura, K. et al. (1988) J. Biochem. 103, 201-208]. Taken together, the results suggest that the pathway from Gg4Cer to IV3α(NeuGcα2-8NeuGc)-Gg4Cer through GM1b(NeuGc) is quite active in mouse immune tissues.View full abstractDownload PDF (2979K) -
Toshiya Sasaki, Kazuko Sukegawa, Michiya Masue, Seiji Fukuda, Shunji T ...1991 Volume 110 Issue 5 Pages 842-846
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA deficiency in α-N-acetylglucosaminidase is known as mucopolysaccharidosis IIIB or Sanfilippo B syndrome. We purified this enzyme almost 39, 000-fold from liver to homo-geneity with 3% recovery. Use of concanavalin A (Con A)-Sepharose and heparin-Sepha-rose resulted in 13.4-fold and 11.6-fold purifications of the enzymatic activity, respectively. The molecular mass was estimated to be 300 kDa by gel filtration and 80 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The isoelectric point was 5.1, optimal pH was 4.5, and the Km for p-nitrophenyl α-N-acetylglucosamine was 0.13-0.20 mM. The purified enzyme was stable at 50°C for 1 h and within the pH range of 6.5-8.5. Anti-serum against the purified enzyme raised in BALB/c mice inhibited the activities of α-N-acetylglucosaminidase.View full abstractDownload PDF (711K)
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