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Volume 128 (2000)
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Volume 128, Issue 4
Displaying 1-20 of 20 articles from this issue
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Marina Martínez-Cayuela, M Carmen García-Pelayo, Ana Lin ...2000 Volume 128 Issue 4 Pages 545-551
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (pahnitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that pahnitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.View full abstractDownload PDF (547K) -
Hiroyuki Iwatsuld, Yi-Min Lu, Kou Yamaguchi, Naoki Ichikawa, Tadao Has ...2000 Volume 128 Issue 4 Pages 553-559
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSYeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F1FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F1FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F1FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.View full abstractDownload PDF (584K) -
Hirokazu Ishida, Tsuyoshi Shirai, Yoshiko Matsuda, Yuji Kato, Masanori ...2000 Volume 128 Issue 4 Pages 561-574
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe computer implementation of a peptide drug-design strategy has been developed. The system is named EmPLiCS (Empirical Peptide Ligand Construction System) according to the strategy of the system, which searches for peptide-ligand structures by referring to empirical rules that are derived from known protein 3D structures. The system was tested on several known peptide-protein complexes. The results demonstrated the ability of this system to detect key residues of peptides that are crucial for interaction with their specific proteins. The system also showed the ability to detect the main chain trace of these peptides. Some of the main chain atoms were detected even though the complete primary structures were not reproduced, suggesting that main chain structure is important in peptide-protein recognition. The results of the present study demonstrated that the empirical rules-based system can generate significant information for use in the design of natural peptide drugs.View full abstractDownload PDF (3099K) -
Tatsuki Kunoh, Takeshi Sakuno, Takakazu Furukawa, Yoshinobu Kaneko, Sa ...2000 Volume 128 Issue 4 Pages 575-584
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhile this Saccharomyces cerevisiae SIN4 gene product is a component of a mediator complex associated with RNA polymerase II, various studies suggest the involvement of Sin4 in the alteration of higher-order chromatin structure. Our previous analysis of a sin4 mutant suggested that the mechanisms of transcriptional repression by Sin4 (mediator) and the Tupl-Ssn6 complex (general repressor) are different. To elucidate the way in which these two repression systems are interrelated, we isolated mutants that exhibit enhanced transcription of a reporter gene harboring the upstream activation sequence (UAS), but still are subject to Tupl-Ssn6-mediated repression. Besides sin4, rgrl, tupI, and ssn6 mutants, we also obtained new mutants that enhance basal transcription even from a core promoter without UAS. Such mutants, designated rbt for regulator of basal transcription, can be classified into at least six complementation groups, i.e., four single (rbtl to rbt4) and two apparently double (rbt5 rbt6 and rbt7 rbt8) mutations. The phenotype of rbt mutants is dependent on the TATA box and not specific to the integration site or kind of core promoter. No significant difference in micrococcal nuclease (MNase) accessibility to the core promoter of test genes was observed between rbt mutants and the wild-type strain, indicating that the higher-order chromatin structure of the core promoter region is not significantly altered in these mutants. The rbtl to rbt4 mutations are suppressed by the Δgain mutation as in the case of the sin4 mutation, but give rise to a different profile from the sin4 mutation with regard to the activity of some of the promoters. From these observations, we suggest that RBT gene product (s) could be novel mediators that act with or in close association with Sin4 but have a function distinct from that of Sin4. Moreover, the fact that rbt mutations nullify Tupl-Ssn6 general repressor-mediated repression is consistent with the idea that the mechanisms of Rbt (mediator)- and Tupl-Ssn6 (general repressor)-mediated repression are interconnected but substantially different.View full abstractDownload PDF (3904K) -
Hiroshi Takagi, Kazunori Hirai, Masaru Wada, Shigeru Nakamori2000 Volume 128 Issue 4 Pages 585-589
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe obtained enhanced thermostability by replacing Ser161 with Cys in subtilisin E from Bacillus subtilis, a cysteine-free alkaline serine protease. The Ser161Cys mutant subtilisin E was purified from the culture supernatant of the recombinant B. subtilis in an oxidizing environment. SDS-polyacrylamide gel electrophoresis and mass spectrometry under oxidizing conditions indicated that the mutant enzyme in part formed an alignmeric protein, which may contain an intermolecular disulfide bond between two surface Cys residues at position 161. Further, no free sulfhydryl groups were detected in the mutant enzyme, suggesting the sulfhydryl modification in a monomeric form under oxidizing conditions. The Ser161Cys mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme. The half-life of thermal inactivation of the mutant was found to be 2-4 times longer than that of the wild-type enzyme. The optimum temperature of the mutant was 55°C, which was 5°C higher than that of the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme reverted to those of the wild-type enzyme. Similar results were obtained for another Cys mutant as to position 194 (wild-type, Ser), which is the same surface residue as Ser161. Possible reasons for the enhanced thermostability of the single-Cys mutant subtilisins E under oxidizing conditions are discussed in terms of two different mechanisms.View full abstractDownload PDF (1150K) -
Koichi Uemttra, Naoki Shibata, Anwaruzzarnan, Masumi Fujiwara, Takash ...2000 Volume 128 Issue 4 Pages 591-599
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMany enzymes are composed of subunits with the identical primary structure. It has been believed that the protein structure of these subunits is the same. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) comprises eight large subunits with the identical amino acid sequence and eight small subunits. Rotation of the side chains of the lysine residues, Lys-21 and Lys-305, in each of the eight large subunits in spinach RuBisCO in two ways produces microheterogeneity among the subunits. These structures are stabilized through hydrogen bonds by water molecules incorporated into the large subunits. This may cause different effects upon catalysis and a hysteretic, timedependent decrease in activity in spinach RuBisCO. Changing the amino acid residues corresponding to Lys-21 and Lys-305 in non-hysteretic Chronzatium vinosum RuBisCO to lysine induces hysteresis and increases the catalytic activity from 8.8 to 15.8 per site per second. This rate is approximately five times higher than that of the higher-plant enzyme.View full abstractDownload PDF (4331K) -
Nobumasa Hara, Masaharu Terashima, Makoto Shimoyama, Mikako Tsuchiya2000 Volume 128 Issue 4 Pages 601-607
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiolindependent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTI: From these observations, we conclude that the Rt6.1 antigen has thioldependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.View full abstractDownload PDF (643K) -
Yoshitaka Ikeda, Souichi Koyota, Hideyuki Ihara, Yukihiro Yamaguchi, H ...2000 Volume 128 Issue 4 Pages 609-619
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic basis of the donor substrate specificity of β, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GIcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower Vmax values, whereas the Km, values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Gle derivatives bind to the enzyme with comparable affinities to those of UDP-GleNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GleNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor.View full abstractDownload PDF (1868K) -
Ayano Matsumoto, Kohdoh Shikata, Fujio Takeuchi, Naoya Kojima, Tsuguo ...2000 Volume 128 Issue 4 Pages 621-628
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RE' activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.View full abstractDownload PDF (1592K) -
Shinya Kamauchi, Toshiaki Fudemoto, Junji Miki, Hiroki Inoue, Hiroshi ...2000 Volume 128 Issue 4 Pages 629-635
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo monoclonal antibodies, β 208 and β 210, against the β subunit of the F1 ATPase from Escherichia coli reacted with an intact β subunit and also a peptide corresponding to a portion of β between residues 1 and 145. Mutations at Ala-1, Val-15, Glu-16, Phe-17, Leu-29, Gly-65, or Leu-66, and His-110 or Arg-111 for β 210 and β 208, respectively, caused decreased antibody binding to β suggesting that these residues form the epitopes and are thought to lie close together on the surface of the β subunit. The topological locations of the corresponding residues in the atomic structure of the bovine β subunit agree well with these expectations, except for Ala-1 and Leu-29. β 210 binds to two β strands including the epitope residues that are 50 residues apart, indicating that this antibody recognizes the tertiary structure of the N-terminal end region. Mutations in the epitope residues of β 210 do not affect the F1 ATPase activity, suggesting that surfaces of the two β strands in the amino-terminal end region are not functionally essential. To analyze the functional importance around His-110 recognized by β 208 we introduced site specific mutations at residues His-110 and Ile-109. Ile-109 to Ala or Arg, and His-110 to Ala or Asp caused defective assembly of F1. However, the His-110 to Arg mutation had no effect on molecular assembly, suggesting that Ile-109 and His-110, especially the positive charge of His-110 are essential for the assembly of F, . The His-110 to Arg mutation caused a large decrease in F1-ATPase activity, suggesting that a subtle change in the topological arrangement of the positive charge of His-110 located on the surface of β plays an important role in the catalytic mechanism of the F1-ATPase.View full abstractDownload PDF (3395K) -
Mitsunori Fultuda, Katsuhiko Mikoshiba2000 Volume 128 Issue 4 Pages 637-645
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSynaptotagmins constitute a family of membrane proteins that are characterized by one transmembrane region and two C2 domains. Recent genetic and biochemical studies have indicated that oligomerization of synaptotagmin (Syt) I is important for expression of function during exocytosis of synaptic vesicles. However, little is known about heterooligomerization in the synaptotagmin family. In this study, we showed that the synaptotagmin family is a type I membrane protein (Nlumen/Ccytoplasm) by introducing an artificial N-glycosylation site at the N-terminal domain, and systematically examined all the possible combinations of hetero-oligomerization among synaptotagmin family proteins (Syts I-XI). We classified the synaptotagmin family into four distinct groups based on differences in Ca2+-dependent and -independent oligomerization activity. Group A Syts (III, V, VI, and X) form strong homo- and hetero-oligomers by disulfide bonds at an N-terminal cysteine motif irrespective of the presence of Ca2+ [Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol.. Chem. 274, 31421-31427]. Group B Syts (I, II, VIII, and XI) show moderate homo-oligomerization irrespective of the presence of Ca2+. Group C synaptotagmins are characterized by weak Ca2+-dependent (Syts IX) or no homo-oligomerization activity (Syt IV). Syt VII (Group D) has unique Ca2+-dependent homooligomerization properties with EC50 values of about 150μM Ca2+ [Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185]. Syts IV, VIII, and XI did not show any apparent hetero-oligomerization activity, but some sets of synaptotagmin isoforms can hetero-oligomerize in a Ca2+-dependent and/or -independent manner. Our data suggest that Ca2+-dependent and -independent hetero-oligomerization of synaptotagmins may create a variety of Ca2+-sensors.View full abstractDownload PDF (2977K) -
Atsushi Mitsumoto, Kwi-Ryeon Kim, Genichiro Oshima, Manabu Kunimoto, K ...2000 Volume 128 Issue 4 Pages 647-654
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.View full abstractDownload PDF (2824K) -
Takeko Kodama, Hiromu Takamatsu, Kei Asai, Naotake Ogasawara, Yoshito ...2000 Volume 128 Issue 4 Pages 655-663
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously reported that YaaH and YrbA are spore proteins of Bacillus subtilis that are required for spore resistance and/or germination and that they have a motif conserved among so-called cell wall binding proteins [Kodama et al. (1999) J. Bacteriol. 181, 4584-4591, Takamatsu et al. (1999) J. Bacteriol. 181, 4986-4994]. In this study, we analyzed the expression of ydhD, ykuD, and ykvP genes, which encode putative proteins containing the same motif. Transcription of ydhD was dependent on SigE, and the raRNA was detectable from 2 h after the cessation of logarithmic growth (T2 of sporulation). ykuD was transcribed by SigK RNA polymerase from T4 of sporulation. Both SigK and GerE were essential for ykvP expression, and this gene was transcribed from T5 of sporulation. Inactivation of these genes by insertion of an erythromycin resistance gene did not affect vegetative growth, spore resistance to heat, chloroform, and lysozyme, or spore germination in the presence of L-alanine or in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride. The His tag fusions of YdhD, YkuD, and YkvP downstream of their natural promoter regions were introduced into a multicopy plasmid. These fusion proteins were produced during sporulation in B. subtilis transformants and were detected in mature spores, indicating that YdhD, YkuD, and YkvP are all proteins intrinsic to spores. Excessive YkuD and YkvP in the sporulating cells did not affect spore resistance or germination. The cells producing excessive YdhD also did not show impaired spore resistance, but their germination properties were changed: the spores revealed reduced response to L-alanine and some of them germinated even without germinants. Escherichia coli β-lactamase, whose signal sequence had been genetically replaced by the cell wall binding motif of YaaH, was produced in sporulating cells, and Western blot analysis indicated that the fused protein was assembled into spores. We speculate that the conserved motif functions as a kind of signal sequence involved in assembly of these proteins on forespores.View full abstractDownload PDF (2820K) -
Masahiko Harata, Yukako Oma, Teruko Tabuchi, Yan Zliang, David J. Stil ...2000 Volume 128 Issue 4 Pages 665-671
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn increasing number of actin-related proteins (Arps), which share the basal structure with skeletal actin but possess distinct functions, have been found in a wide variety of organisms. Individual Arps of Saccharomyces cerevisiae were classified into Arps 1-10 based on the relatedness of their sequences and functions, where Arpl is the most similar to actin, and ArplO is the least similar. While Arps 1-3 and their orthologs in other organisms are localized exclusively in the cytoplasm, Arp4 (also known as Act3) is localized in the nucleus and is involved in transcriptional regulation. Here we examined the more divergent Arps for possible nuclear functions. We show that Arps 5-9 are localized in the nucleus, but Arp10 is not. The nuclear export signals identified in actin are well conserved in the cytoplasmic Arps, Arps 1-3, but less conserved in the nuclear Arps. Gel filtration chromatography experiments show that the nuclear Arps are larger than monomer in size and thus are present in multi-protein complexes. Since nuclear protein complexes containing Arps are found to be responsible for histone acetylation and chromatin remodeling, it is suggested that most of the divergent Arps are involved in the transcriptional regulation through chromatin modulation.View full abstractDownload PDF (3771K) -
Kiyoshi Ito, Takahiko Inoue, Tsutomu Kabashima, Naota Kanada, Hua-Shan ...2000 Volume 128 Issue 4 Pages 673-678
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMolecular cloning of the gene and the crystal structure of the prolyl aminopeptidase [EC 3. 4. 11. 5] from Serratia nmrcescens have been studied by us [J. Bioehem. 122, 601-605 (1997); J. Bioehem. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (kcat/Km), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-βNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the SI site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.View full abstractDownload PDF (2646K) -
Kwang-Hwan Jhee, Tohru Yoshimura, Edith Wilson Miles, Sou Takeda, Ikuk ...2000 Volume 128 Issue 4 Pages 679-686
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria. The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase. In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate. L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination. The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate. Aminodeoxychorismate lyase is identical to Damino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer. Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction. The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, 0-amino acid aminotransferase and branched-chain L-amino acid aminotransferase [Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K. -H., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 128, 29-38].View full abstractDownload PDF (695K) -
Shinsaku Maruta, Tomoki Aihara, Yasuo Uyehara, Kazuaki Homma, Yasunobu ...2000 Volume 128 Issue 4 Pages 687-694
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the presence of excess amounts of fluorine, a physiological divalent cation, magnesium (Mg2+), forms a novel phosphate analogue, magnesium fluoride (MgFn). Park et al. [Biochim. Biophys. Acta 1430, 127-140 (1999)] previously demonstrated that MgADP•MgFn forms a complex with myosin subfragment-1 (S-1), and the S-1, ADP•gFn ternary complex mimics a transient state in the activity cycle of ATPase. In the present study, localized conformations in the regions of highly reactive cysteine and lysine residues, Cys 707 (SH1), Cys 697 (SH2), and Lys 83 (RLR), which change their conformations markedly during ATP hydrolysis, were studied using fluorescent probes and chemical modification. The global shape of the complex was also studied using small angle X-ray solution scattering and compared it with other previously reported myosin-ADP•fluorometal ternary complexes. The results suggest that the overall conformation and localized functional regions of the complex are quite similar to those in the presence of ATP, indicating that the complex mimics the M¨•ADP•Pi steady state.View full abstractDownload PDF (1396K) -
Shinsaku Maruta, Kazuaki Homma2000 Volume 128 Issue 4 Pages 695-704
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin has three highly-conserved, unique loops [B (320-327), M (677-689), and N (127-136)] at the entrance of the ATP binding cleft, and we previously showed that the effects of actin are mediated by a conformational change in loop M [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, loops M and N were photolabeled respectively with fluorescent probes Mant-8-N3-ADP and Mant-2-N3-ADP in order to study conformational changes in the loops related to energy transduction. The effect of actin on the conformation of loop N was examined by analyzing fluorescence polarization and acrylamide quenching; the results were then compared with those previously reported for loop M. In contrast to loop M, the fluorescence polarization and the value of K6V of the Mant-groups crosslinked to loop N were slightly affected by actin binding. To study conformational changes in loops M and N during the ATPase cycle, FRET was analyzed using TNP-ADP•Fn and TNP-ADP•AIF-4 as FRET acceptors of Mant fluorescence. The resultant estimated distances between loop M and the active site differed for the Mant-Sl•TNP-ADP•BeFn and Mant-S1•TNP-ADP•AIF-4 complexes, whereas the distances between loop N and the active site differed slightly. These findings indicate that the conformation of loop M changes during the ATPase cycle, suggesting that Loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites. Loop N, by contrast, is not significantly flexible.View full abstractDownload PDF (1928K) -
Kazuhisa Suehiro, Jun Mizuguchi, Kiyoto Nishiyama, Sadaaki Iwanaga, Da ...2000 Volume 128 Issue 4 Pages 705-710
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin α5β1 is a fibrinogen receptor on endothelial cells [Suehiro, K, Gailit, J., and Plow, E. F. (1997) J. Biol. Chem, 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified α5β1 and have identified the recognition sequence in fibrinogen for α5β1. The binding of fibrinogen to immobilized α5β1 was selectively supported by Mn2+. Fibrinogen bound to purified α5β1 in a time-dependent, specific, and saturable manner in the presence of Mn2+, and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-α5 and anti-α5β1 monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aα572-574 significantly inhibited the binding of fibrinogen to α5β1, whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aα95-97 or the C-terminus of the γ-chain did not. Furthermore, substituting RGE for RGD at position Aα95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aα572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aα572-574 is required for the interaction of fibrinogen with α5β1.View full abstractDownload PDF (556K) -
Ayako Kobayashi, Mild Kasano, Tatsuo Maeda, Shin-ichiro Hort, Kiyoto M ...2000 Volume 128 Issue 4 Pages 711-718
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTAPL is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that is transcribed in various rat tissues [Yamaguchi, Y., Kasano, M., Terada, T., Sato, R., and Maeda, M. (1999) FEBS Lett. 457, 231-236]. Primary structures of the human and mouse orthologous counterparts were deduced from cDNAs cloned by means of polymerase chain reaction, and they were compared with that of the rat. The mammalian TAPLs (rat, mouse, and human) are highly conserved, since about 95% of the amino acid residues are identical between rodents and man. Phylogenetic analysis demonstrated that the evolutional rate of TAPL is much slower than those of TAP1 and TAP2, although TAPL could have diverged from an ancestor of TAP1 or that of TAP1 and TAP2. The TAPL-GFP fusion protein transiently expressed in Cos-1 cells was co-localized with PDI, suggesting that TAPL is inserted into endoplasmic reticulum membrane. The conservation of the peptide-binding motifs of TAP proteins in TAPL raises the possibility that the TAPL might be a peptide transporter. The gene for human TAPL is assigned to chromosome 12q24.31-q24.32, while those for TAP1 and TAP2 are located at the MHC locus of chromosome 6p21.3. Furthermore, the transcription of TAPL gene is not responsive to interferon-γ, in contrast to TAP1 and TAP2. These results indicate that the gene regulation of TAPL is different from those of TAP1 and TAP2.View full abstractDownload PDF (3004K)
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