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Volume 113, Issue 1
Displaying 1-22 of 22 articles from this issue
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Shoji Ogiwara, Hiroyoshi Hidaka, Takashi Sugimoto, Ryo Teradaira, Keis ...1993 Volume 113 Issue 1 Pages 1-3
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe concentrations of oncopterin, N2-(3-aminopropyl) biopterin, a new pterin compound, were determined in urine from various cancer patients by HPLC on a reverse-phase or ion-exchange column. The concentration of oncopterin increased after acid hydrolysis, indicating that it exists as an amide in urine. The oncopterin concentrations were very low in the urine of healthy controls. Among the urine samples examined, those from cases of solid cancers, e. g., hepatomas, prostatic cancer and bladder cancer, exhibited very high levels; and those from cases of blood cancers, e. g., myelomats, acute myelocytic leukemia, and lymphomas, showed moderate increases. Oncopterin may thus be a new biochemical marker of some types of cancer.View full abstractDownload PDF (267K) -
Satoshi Akiba, Takashi Sato, Tatsuzo Fujii1993 Volume 113 Issue 1 Pages 4-6
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFollowing stimulation of rabbit platelets with thrombin, phospholipase A2 (PLA2) activity increased in the Triton X-100-insoluble residue. Although the PLA2 activity was dependent on the protein content of the residue from the stimulated cells, the specific activity was higher than that in the case of unstimulated cells. The enzyme activity was inhibited by p-bromophenacyl bromide and increased significantly with 0.5-10 μM Ca2+. The enzyme hydrolyzed phospholipids having an arachidonoyl residue more effectively than ones with a linoleoyl residue. In addition, 70% of the enzyme activity was immunoprecipitated with a monoclonal antibody against cytosolic PLA2 of rabbit platelets, while it was inhibited by only 20% by an antibody that neutralizes the activity of group IIPLA2. These results suggest an increase in the association of cytosolic PLA2 with the cytoskeleton upon stimulation of rabbit platelets.View full abstractDownload PDF (349K) -
Tomohiko Fukuda, Yoshio Imai, Masavuki Komori, Masahiko Nakamura, Emi ...1993 Volume 113 Issue 1 Pages 7-12
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThreonine-303 of rabbit P450 2E1, 3 which is putatively located at the distal heme surface, was replaced by serine and valine via site-directed mutagenesis. In the oxidized state, the Ser-mutated P450 exhibited a low- and high-spin mixed-type (low⟩high) absorption spectrum, whereas the Val-mutated P450, like the wild-type P450, exhibited a nearly high-spin type spectrum. The reduced CO complexes of the Ser- and Val-mutated P450s, as well as that of the wild-type P450, showed a Soret absorption maximum at 452 nm. Both mutated P450s were active in the hydroxylation of C10 to C18 fatty acids at somewhat lower rates than the wild-type P450. The Val-mutated P450 gave the same two products (the major one is probably the ω-1 hydroxy analog) as the wild-type P450, while additional products were formed on incubation with C11 to C17 fatty acids as substrates of the Ser-mutated P450; a total of four products was detected for each of the C12 to C15 fatty acids, and three for each of the C11, C16, and C17 homologues. The metabolites of laurate were determined by GC-MS analysis to be the ω-1, ω-2, ω-3, and ω-4 hydroxy counterparts. The Ser-mutated P450 hydroxylated drug substrates at almost the same rates as the wild-type P450, while the mutation to valine significantly lowered the drug hydroxylase activities. These findings indicate that Thr-303 of P450 2E1 plays an important role in determining its substrate specificity (hydroxylation position), probably by fixing the fatty acid substrate at the proper position on the P450 protein through the interaction between the γ-methyl group of Thr-303 and the side chain of the fatty acid.View full abstractDownload PDF (716K) -
Shuji Hada, Shinobu Fujii, Seiji Inoue, Kiyoshi Ikeda, Keizo Teshima1993 Volume 113 Issue 1 Pages 13-18
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInitial velocity data for the hydrolysis of micellar 1, 2-diheptanoyl-sn-glycero-3-phos-phorylcholine (diC7PC) catalyzed by bovine pancreatic PLA2 (Group I) were analyzed using the Michaelis-Menten equation. The Km value for the micellar substrate was found to be independent of Ca2+ concentration, as was the Km value for the monodispersed substrate. The pH dependence curve of Km in the presence of saturating amounts of Ca2+ showed two transitions reflecting large pK shifts of two ionizable groups from 5.0 to 5.45 and from 9.5 to 10.25, whereas the Km value for the monodispersed substrate was independent of pH [Fujii et al. (1991) J. Biochem. 110, 1008-1015]. The pH dependence curve of kcat showed three transitions, indicating the participation of three ionizable groups with pK values of 5.45, 8.4, and 10.25. Deprotonation of the first group and protonation of the third group were found to be essential for catalysis. The respective groups were assigned as the catalytic group His 48, the N-terminal α-amino group, and invariant Tyr 52. The present results as well as those foranotherGroup I PLA2 (Naja naja atra) are very different from those for Group II PLA2s (Agkistrodon halys blomhofi and Trimeresurus flavoviridis), which showed Ca2+-dependent substrate binding and no participation of the α-amino group in catalysis [Teshima et al. (1989) J. Biochem. 106, 518-527; Nishimura et al. (1992) J. Biochem. 111, 210-218]. Although the catalytic efficiency of the bovine enzyme is very low as compared with those of snake enzymes, the present results confirmed that this enzyme shows properties characteristic of Group I enzymes as described above, indicating that no special catalytic mechanism needs to be assumed for the bovine pancreatic enzyme.View full abstractDownload PDF (739K) -
Masaru Tanokura, Setsuro Ebashi1993 Volume 113 Issue 1 Pages 19-21
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin subfragment-1 pyrophosphate (S1•PP1) complex and S1•ADP complex were observed with 31P NMR at various temperatures between 0 and 25°C. The signal of S1•PP1 complex showed a small temperature dependence, indicating that a single conformation exists for the complex. It also showed that the electron density around phosphorus nuclei was increased upon the formation of complex. On the other hand, the signal of S1•ADP complex was clearly dependent on temperature and indicated the presence of two forms, i.e., high-temperature and low-temperature forms. In the high-temperature form, the electron density around β-phosphate was decreased upon the formation of complex with Sl. In the low-temperature form, the distribution of electrons around phosphorus nuclei is extremely anisotropic due to the tight interaction of S1 and the phosphate moieties of ADP.View full abstractDownload PDF (351K) -
Ariel Fernández1993 Volume 113 Issue 1 Pages 22-28
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMaking use of parallel Monte Carlo simulations of competing folding pathways, we determine the specific stages in the in vivo sequential folding of group I pre-mRNA transcripts where participation of a trans-acting factor or an already-assembled portion of the transcript itself is required to generate a catalytically competent structure and subsequently shape the 3' splicing site. Thus, the model for ribozyme function revealed by the simulations should be regarded as dynamical since it is based on sequential folding. Our main aim is to prove that sequential folding warrants the chronology of splicing events required for ribozyme function, a feature which cannot be reproduced in existing static models of folding based on free energy minimization. The effect of trans-acting factors on the catalytically relevant folding pathway is assessed by comparing the in vitro folding pathway with the pathway that leads to the structure that splices the 5' extremity. The latter has been inferred previously by other authors using phylogenetic analysis. Since our model is rooted in multiprocessed folding algorithms, we concentrate on mitochondrial pre-mRNA transcripts belonging to group I which undergo no detectable self-splicing in vitro. As an illustrative example, the results have been specialized to the fourth intron of the yeast apocytochrome b gene (YCOB4). A crucial feature of our approach, irreproducible in previous models, is that it accounts for a meaningful scenario of competition between hydrolysis at the 3' extremity of the intron and exon-exon ligation. We prove that this scenario explains how the premature formation of conserved helix P10 is prevented until 5' cleavage has taken place. Two experiments based on site-directed mutagenesis are proposed to test the predictions in the case of YCOB4.View full abstractDownload PDF (883K) -
Toshinaga Maeda, Rieko Oyama, Koiti Titani, Kiyotoshi Sekiguchi1993 Volume 113 Issue 1 Pages 29-35
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFibronectin contains at least two distinct oligopeptide sequences serving as signals for the interaction with cell surface adhesion receptors termed integrins. One of these sequences, Arg-Gly-Asp-Ser (RGDS) tetrapeptide, was shown to be transferred to a truncated form of Staphylococcal IgG-binding protein (hereafter referred to as tSPA) with retention of its cell-adhesive activity [Maeda, T. et al. (1989) J. Biol. Chem. 264, 15165-15168]. We have extended the observation to another cell-adhesive sequence, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (referred to as“CS1” sequence), to demonstrate that i) the tSPA grafted with the sequence mediated adhesion of human lymphoma and rhabdomyosarcoma cells, mouse melanoma cells, but not of hamster fibroblasts; ii) antibodies against integrin α4 and β1 subunits specifically inhibited cell adhesion mediated by the CS1-grafted tSPA; iii) a heterodivalent tSPA grafted with both RGDS and CS1 sequences at different sites was more potent in promoting cell adhesion than the monovalent tSPAs grafted with either sequence alone. These results indicate that not only the RGDS but also the CS1 sequence can be transferred to tSPA with retention of its cell-adhesive activity as well as its cell-type specificity, and that the grafted CS1 sequence is recognized by the same integrin isotype as the authentic sequence within intact fibronectin.View full abstractDownload PDF (4211K) -
Yukio Mishima, Tetsuji Nishimura, Masami Muramatsu, Ryo Kominami1993 Volume 113 Issue 1 Pages 36-42
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSS100 extract prepared from rapidly growing mouse FM3A cells (approx.5×105 cells/ml) transcribed ribosomal RNA gene (rDNA) much more actively in vitro than that from stationary phase cells (1-2×106 cells/ml). When the inactive S100 extract was preincubated with NAD+, rDNA transcriptional activity was restored almost to the level of the active extract. The extract activated with NAD+ exhibited a gel-shift band in the gel mobility shift assay and enhancement of protection of the sequence between -44 and -8 nt from the initiation site from exonuclease III digestion. Such an extract labeled with [32P]NAD+ was analyzed by immunoprecipitation with anti-RNA polymerase I (pol I) antibody; a protein with Mr 130 kDa was detected. In contrast, the polypeptide was hardly labeled in the active extract. 3-Aminobenzamide, a specific inhibitor of poly ADP-ribosylation, did not inhibit the activation by NAD+. These results suggest that the activation by NAD+ is due to enhancement of the formation of initiation complex by mono ADP-ribosylation of the second-largest subunit (130 kDa) of pol I.View full abstractDownload PDF (4514K) -
Sanae Kato, Kunihiko Konno1993 Volume 113 Issue 1 Pages 43-47
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubstructure of the myosin rod and its correlation to the filament formation were inves-tigated by using fish myosin rod. It was found that fish rod contains a unique chymotrypsin susceptible site, 40 kDa from the COOH-terminus or 20 kDa downstream from the subfragment-2/light meromyosin junction (S-2/LMM junction). Cleavage at this new site produced subfragment-2 possessing 95 kDa subunit (95k S-2) and light meromyosin possessing 40 kDa subunit (40k LMM). The latter is the shortest unit ever reported to exhibit filament formation. Moreover, the 40k LMM was able to form filaments indepen-dently of the presence of Mg2+, while filament formation of rod and ordinary LMM (70k LMM) was promoted by Mgt2+ addition. These results indicated that the Mg2+ binding sites are present within the NH2-terminal 20 kDa region of the 70k LMM. We concluded that the COOH-terminal 40 kDa portion of rod is responsible for the self-assembly ability of myosin, while the NH2, -terminal 20 kDa region of the 70k LMM is the regulatory domain for thick filament formation through Mg2+-binding.View full abstractDownload PDF (3079K) -
Taketomo Fujiwara, Yoshihiro Fukumori, Tateo Yamanaka1993 Volume 113 Issue 1 Pages 48-54
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour different membrane-bound b-type cytochromes were found to occur in Halobacterium halobium strain L-33, and two of them, b-558 and b-562, were purified to homogeneity. Cytochrome b-558 showed absorption peaks at 414 and 526 nm in the oxidized form, and peaks at 425, 528, and 558 urn in the reduced form. Its α peak at 558 nm in the reduced form was asymmetric with a shoulder at around 554 nm. At liquid nitrogen temperature, the α peak was split into two peaks at 549 and 556 nm which appeared to be the α peaks of cytochromes c and b, respectively. The cytochrome contained 1 mol of protoheme in 28, 500g, and was composed of one molecule each of two subunits with molecular masses of 15.4 and 11.7 kDa, respectively. The heme seemed bound to the larger subunit. The cytochrome was very autoxidizable and its redox potential at pH 8.0 was -75 mV. Cytochrome b-562 showed absorption peaks at 417 and 530 nm in the oxidized form and peaks at 431, 531, and 562 nm in the reduced form. The cytochrome was composed of only one polypeptide (25kDa) and seemed to contain one protoheme molecule per molecule.View full abstractDownload PDF (1777K) -
Yuji Goto, Eiichi Kogure, Toshio Takagi, Saburo Aimoto, Takaaki Aoba1993 Volume 113 Issue 1 Pages 55-60
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCircular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90°C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25°C. It was tentatively concluded that the N-terminal region contains b'-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.View full abstractDownload PDF (719K) -
Kazuya Murakami, Yasumitsu Takagi, Katsuyoshi Mihara, Tsuneo Omura1993 Volume 113 Issue 1 Pages 61-66
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is generally believed that liver carboxyesterases are localized exclusively in the endoplasmic reticulum (ER), mostly in the lumen, loosely bound to the inner side of the membrane. A cDNA clone, clone (8-1/2-1) supposed to code for one of the isozymes, carboxyesterase E1, was isolated by Takagi et al. [J. Biochem. 104, 801-806 (1988)]. However, the protein coded by clone (8-1/2-1) had no consensus ER retention signal at its carboxy terminus, and the mechanism of its retention by ER lumen was unclear. When clone (8-1/2-1) was expressed in COS cells in this study, the plasmid-coded protein was secreted into the medium. When the carboxy terminal portion of the clone (8-1/2-1)-coded protein was replaced with the corresponding region of another carboxyesterase, pI 6.1 esterase, which had the HVEL sequence at the carboxy terminus, the chimeric protein was retained in the COS cells. We searched for a secretory form carboxyesterase in rat blood immunochemically using polyclonal antibodies to carboxyesterase E1, and detected a crossreacting protein with a molecular weight of 68 kDa. The molecular weight was decreased by endoglycosidase F treatment but not by endoglycosidase H treatment, indicating that the protein carries complex type sugar chains. In addition, the cross-reacting protein was labeled with [3H] diisopropylfluorophosphate (DFP), suggesting that the protein has an esterase-type active center serine. We confirmed that the antibodies against the carboxy-terminal portion (CNPPQTEHTEHT-COOH) of the clone (8-1/2-1)-coded protein reacted only with the blood protein, whereas the antibodies against the carboxy terminal portion (CAEEPSHWKHVEL-COOH) of pI 6.1 esterase reacted only with the microsomes. We thus conclude that the isozyme of liver carboxyesterases coded by clone (8-1/2-1) is not carboxyesterase El but a secretory form carboxyesterase, which has not been noticed so far. The physiological significance of the secretory form carboxyesterase, carboxyesterase See, in the blood remains to be clarified.View full abstractDownload PDF (3367K) -
Tadashi Yoshimoto, Taiji Shimoda, Ana Kitazono, Tsutomu Kabashima, Kiv ...1993 Volume 113 Issue 1 Pages 67-73
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pyroglutamyl peptidase [EC 3. 4. 11. 8] gene from Bacillus amyloliquefaciens was cloned and expressed in Escherichia coli DH1. The transformant of E. coli DH1 harboring plasmid pBPG 1 with a 2.1 kb chromosomal DNA fragment showed 80-fold higher activity than B. amyloliquefaciens. The nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chaintermination method. An open reading frame of 648 bp starting with an ATG methionine codon was found, which encodes a protein of 215 amino acid residues with a deduced molecular weight of 23, 286. The enzyme has two cysteine residues (Cys68 and Cys144) per subunit molecule. Substitution of Cys144 with Ser by site-directed mutagenesis resulted in a complete loss of the activity, while that of Cys68 with Ser did not affect the activity at all. This result and titration with DTNB suggest that Cys144 is concerned in the catalytic action and Cys68 is located inside the enzyme. The expressed enzyme was purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with an activity recovery of 42.7%. The enzyme was most active at pH 6.5 and stable at pH 7.0-9.0. Its molecular weight was estimated to be 51, 000 by gel filtration, suggesting it to be a dimer. Big crystals of the wild and PCMB-modified enzymes were obtained by the hanging drop method.View full abstractDownload PDF (4402K) -
Kunihiko Gekko, Keigo Yamagami, Yuichi Kunori, Shigeyuki Ichihara, Mic ...1993 Volume 113 Issue 1 Pages 74-80
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15°C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2°C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km was not influenced but kcat was greatly decreased by these mutations, resulting in 240-and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction.View full abstractDownload PDF (870K) -
Kazuya Yamanishi, Zafar I. Randawa, Dennis Brown, Yoshihiro Masui, Set ...1993 Volume 113 Issue 1 Pages 81-87
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor ([3-153]M-CSF) consists of 302 amino acid residues and has a molecular mass of about 32 kDa, as estimated by SDS-PAGE. Two covalently linked subunits constitute a bioactive homodimer. The structure of the purified protein, expressed in Escherichia coli and refolded from inclusion bodies, was studied. The amino acid sequence was determined by automated Edman degradation of fragments obtained from degradation with CNBr and iodosobenzoic acid as well as by digestion with Glu-C endopeptidase of reduced and alkylated M-CSF. The absence of free thiol groups in the molecule was confirmed with Ellman reagent, which indicated the presence of seven disulfide linkages per homodimer. Sequence analysis of cystine-containing peptides, identified by comparing the peptide maps from unmodified and performic acid-oxidized pepsin digests, gave the following results. (1) Six out of seven disulfide linkages were formed between Cys 7 and Cys 90, Cys 48 and Cys 139, and Cys 102 and Cys 146 at each pair of positions as either intra- or inter-chain disulfides. (2) The remaining disulfide linkage linked Cys 31 of one subunit to Cys 31 of the second subunit of M-CSF. Based on our findings, a two-dimensional model is proposed in which the possible covalent linkage is suggested between the two subunits of the bioactive [3-153]M-CSF molecule.View full abstractDownload PDF (1110K) -
Naoki Kimura, Akiko Fukushima, Kinichiro Oda, Susumu Nakada1993 Volume 113 Issue 1 Pages 88-92
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new in vivo replication system for influenza virus was developed by using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein genes can be expressed in response to dexamethasone. The chimeric NS-chloramphenicol acetyltransferase (CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of the influenza virus RNA segment 8 can be replicated [both genomic RNA (vRNA) and complementary RNA (cRNA) were transcribed] in the clone 76 cells treated with dexamethasone. These data indicate that three RNA polymerase proteins (PB1, PB2, and PA) and nucleoprotein are sufficient for replication of the influenza virus genome. Analysis of mutant cRNAs containing a base-substitution or a deletion in the 3'-conserved terminal 13 nucleotides revealed that important cis elements in the cRNA for vRNA synthesis reside at positions 2, 3, and 7 to 13 nucleotides from the 3'-end.View full abstractDownload PDF (1862K) -
Hiroyuki Iwamoto, Masayuki Ohmori, Masahiro Ohno, Junzo Hirose, Keitar ...1993 Volume 113 Issue 1 Pages 93-96
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction between pullulanase from Klebsiella pneumoniae and α-, β-, and γ-cyclodextrins and 6-O-α-glucosyl-α-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin was examined by means of inhibition studies of the enzyme activity, UV difference spectroscopy, and flow calorimetry. All the above cyclodextrins were found to be competitive inhibitors, but β-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin showed strong inhibition, the inhibitor constants being two orders of magnitude less than those of α- and γ-cyclodextrins. The difference spectra of β-cyclodextrin were slightly but significantly different from those of the other cyclodextrins, showing blue shift of a few nanometers. Moreover, only β-cyclodextrin has a positive entropy change upon binding with the enzyme; all the other cyclodextrins have negative values. These results show that the binding mode of β-cyclodextrin is subtly different from those of α- and γ-cyclodextrins.View full abstractDownload PDF (467K) -
Fumio Watanabe, Yoshihisa Nakano, Yoshiyuki Tamura, Erhard Stupperich1993 Volume 113 Issue 1 Pages 97-100
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the corrinoid specificity of the cytosolic cobalamin-binding protein of Euglena gracilis, inhibition of the binding of radioactive cyanocobalamin to the cytosolic binding protein was studied with a variety of cobalamin analogues. The cytosolic cobalamin-binding protein showed an absolute requirement for the α-axial ligand (the cobaltcoordinated nucleotide) in cobalamin binding, but was not able to recognize certain differences in the base or ribose moiety. Regarding the contributions of the b-, d-, and e-propionamide side chains in the binding of cobalamin to the cytosolic protein, the order of the contributions was shown to be b>d>e; in particular the b-propionamide side chain was essential for the formation of the protein-cobalamin complex. No involvement of the β-axial ligand or the alkanolamine group in the binding of cobalamin to the protein was found.View full abstractDownload PDF (450K) -
Daisuke Tsuru, Seiichi Imajo, Shin-ya Morikawa, Tadashi Yoshimoto, Mas ...1993 Volume 113 Issue 1 Pages 101-105
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active site structure of the Zn-containing neutral protease from Bacillus subtilis var. amylosacchariticus (BAND) was predicted by computer-aided modeling on the basis of the three-dimensional structure of thermolysin (TLN). As expected from the high homology in amino acid sequence of the two enzymes, the overall folding of BANP was very similar to that of TLN. Glu144, Tyr158, and His228 of BANP were located near the active site Zn ion, to which three amino acid residues, His143, His147, and Glu167, were coordinated. This model is supported by the previous results that chemical modifications of Tyr158 and photooxidation of His228 of BANP markedly affect the proteolytic activity of the enzyme. Interestingly, BANP was found to be significantly less sensitive to metalloprotease inhibitors such as phosphoramidon and talopeptin. From a comparison of the enzyme-inhibitor complex models between BANP and thermolysin, it is suggested that replacement of Thr129 in TLN by Phe130 in BANP is related to difference in inhibitor sensitivity between BANP and TLN.View full abstractDownload PDF (469K) -
Retsu Miura, Yasuzo Nishina, Kyosuke Sato, Shigeru Fujii, Kiyo Kuroda, ...1993 Volume 113 Issue 1 Pages 106-113
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 13C- and 15N-NMR spectra of porcine kidney medium-chain acyl-CoA dehydrogenase (MCAD) reconstituted with 13C- and 15N-enriched FADs were measured. The positions of selective enrichment were C(2), C(4), C(4a), C(10a), N(1), N(3), and N(5) of the isoalloxazine nucleus of FAD. The NMR signals of the labeled atoms were observed as broad but distinct peaks in each NMR spectrum. The chemical shift values of the 2-, 4-, 4a-, and 10a-13C for the oxidized form of MCAD were 159.5, 166.8, 141.1, and 155.5 ppm, respectively, relative to the methyl resonance of 3-(trimethylsilyl)propionic acid-d4, while those of 1-, 3-, and 5-15N for the oxidized form were 183.6, 161.1, and 334.7 ppm, relative to liquid ammonia, respectively. The upfield shift of 2-13C of MCAD relative to that of FMN in the aqueous medium and its downfield shift relative to that of tetraacetylriboflavin in an apolar medium imply that a weaker hydrogen bond exists between C(2)=O and apoMCAD or a water molecule than that of free FMN with a water molecule. That the 4-13C resonance was observed downfieldshifted relative to that of free FMN in aqueous solution suggests a strong hydrogen bond between C(4)=O and apoMCAD. The chemical shift for 4a-13C in oxidized MCAD is considerably downfield-shifted from that of FMN or any other flavoprotein observed thus far, indicating a unique environment around this position in MCAD. The 1-15N resonance of MCAD was most upfield-shifted among the flavoproteins observed. This indicates that a strong hydrogen bond is formed between N(1) of flavin and the protein or a water molecule. The slight downfield shift of 3-15N and the similar chemical shift value for 5-15N relative to FMN in aqueous solution also indicate the presence of strong hydrogen bonds at these positions with the protein. In the reduced form of the enzyme, the chemical shift values for 2-, 4-, 4a-, and 10a-13C and 1-, 3-, and 5-15N were 160.3, 160.6, 106.1, 155.7, 175.1, 151.4, and 63.4 ppm, respectively. From a comparison of these chemical shift values of the reduced form with those for neutral and anionic forms of reduced FMN, it was concluded that FAD in the reduced state of MCAD is anionic at N(1) of the flavin moiety. Among the chemical shift values for reduced MCAD, that of 4a-13C is particularly downfield-shifted from that of anionic reduced FMN. This indicates lower electron density at this position and is consistent with the low reactivity of the reduced enzyme toward molecular oxygen. In the 13C- and 15N-NMR spectra of the complex of MCAD with a substrate analog, acetoacetyl-CoA, the 5-15N resonance was specifically upfield-shifted from that of the free oxidized MCAD, while other resonances were not appreciably shifted. This suggests strongly that this substrate analog, and hence a substrate, induces a specific change in the electronic state at N(5) of FAD in the substrate (analog)-MCAD complex.View full abstractDownload PDF (1039K) -
Takashi Kaneko, Yoshitaka Iba, Koichi Zenita, Katsuyoshi Shigeta, Koic ...1993 Volume 113 Issue 1 Pages 114-117
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStage-specific embryonic antigen-1 (SSEA-1) is a well-known carbohydrate antigen that is specifically expressed on the surface of cancer cells as well as embryonic cells. In this study, starting with a previously established hybridoma producing a monoclonal antibody (named H18A) to Lewis Y antigen, which is closely related to SSEA-1, we cloned genomic DNA encoding active variable regions both of heavy and light chains of the antibody. Sequence analysis showed that VH and VK genes of H18A were in the VH7183 family and VKC1 family, respectively. A transfected cell line named HC-H18A-7 expressing a recombinant chimeric H18A composed of mouse-derived antigen-binding variable regions and humanderived constant regions was established. The chimeric H18A was purified to homogeneity and shown to bind purified Lewis Y antigen with the same dose-response curve as the original H18A. The chimeric H18A looks more promising for clinical application than the original mouse-derived H18A because its antigenicity is expected to be reduced.View full abstractDownload PDF (410K) -
Hideo Katoh, Shinji Satomura, Shuji Matsuura1993 Volume 113 Issue 1 Pages 118-122
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and rapid analytical method for the structural identification of sugar chains involving lectins and ultra-filtration was developed. In the procedure, a mixture of fluorogenic sugar chains is mixed with an unmodified lectin. Unbound sugar chains are removed by ultrafiltration. Qualitative differences between sugar chains from the sample and from the filtrate were determined by high performance liquid chromatography. The structures of different sugar chains could be determined from the specificity of the lectins used. Because the lectins used for the procedure were not modified, both the lectins used and sugar chains could be recovered. The method is simple and can be used for structural analysis of sugar chains.View full abstractDownload PDF (443K)
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