All issues
Volume 129 (2001)
- Issue 6 Pages 851-
- Issue 5 Pages 653-
- Issue 4 Pages 491-
- Issue 3 Pages 335-
- Issue 2 Pages 185-
- Issue 1 Pages 1-
Volume 129, Issue 5
Displaying 1-26 of 26 articles from this issue
- |<
- <
- 1
- >
- >|
-
Hiroyuki Sorimachi, Koichi Suzuki2001 Volume 129 Issue 5 Pages 653-664
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecent very rapid developments in genome and EST projects have identified an increasing number of gene products homologous to those that were previously identified by other methods. Calpain is no exception. At the time this review is written, 83 genes from 23 living organisms have been identified in the database to encode amino acid sequences showing significant similarities to the protease domain of “conventional” calpain, which was first purified as a homogeneous protein in 1978. Progress in genome/EST projects has occurred so quickly that there seems to be some confusion as to the identity of each calpain molecule. This review will attempt to clarify all calpain homologues, to describe the common and differing features of calpain homologues in terms of structure-function relationship, and to discuss the evolutionary process of calpain.View full abstractDownload PDF (6109K) -
Yuriko Kobayashi, Shunsuke Arai, Keizo Waku, Takayuki Sugiura2001 Volume 129 Issue 5 Pages 665-669
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS2-Arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p42/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with 2-AG. The addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells prior to the addition of 2-AG abolished the response induced by 2-AG, indicating that the CB2 receptor is involved in the response. G protein Gi or Go is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by 2-AG. CP55940 and anandamide also induced the activation of p42/44 MAP kinase, although the activation by anandamide was less pronounced than that by 2-AG or CP55940. These results suggest that 2-AG may play an important physiological role in this type of cell through the activation of the p42/44 MAP kinase cascade.View full abstractDownload PDF (1276K) -
Hashim Elmileik, Takanori Kumagai, Manuel Berengena, Kazuhiro Ueda, Ma ...2001 Volume 129 Issue 5 Pages 671-674
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAddition of bleomycin (Bm) to an NIH/3T3 cell culture induced the overproduction of four cellular proteins [Kumagai and Sugiyama (1998) J. Biochem. 124, 835-841]. The two proteins were identified on N-terminal amino acid sequence analysis as calreticulin and mitochondrial matrix protein P1, which are known as heat shock proteins, respectively. In this study, we cloned the calreticulin promoter region from the genomic DNA of NIH/3T3 cells and observed that heat shock treatment at 42°C or the addition of Bm to the cell culture caused overexpression of the luciferase gene controlled by the cloned calreticulin promoter. This suggests that Bm induces the transcriptional activation of stress-heat shock genes. We constructed an expression vector for mammalian cells, which is controlled by the calreticulin promoter.View full abstractDownload PDF (1603K) -
Hiroshi Maeda, Osamu Mizutani, Youhei Yamagata, Eiji Ichishima, Tasuku ...2001 Volume 129 Issue 5 Pages 675-682
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule.View full abstractDownload PDF (1837K) -
Akinobu Hino, Makiko Morita, Mizuho Une, Kingo Fujimura, Taiju Kuramot ...2001 Volume 129 Issue 5 Pages 683-689
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied the effects of deoxycholic acid and its three epimers with β-hydroxyl groups (3α, 12β-, 3β, 12α-, and 3β, 12β-dihydroxy-5β-cholan-24-oic acids), which were hydrophilic and less cytotoxic, on lipid peroxidation to elucidate the relationship between structural features of bile acids and their effect on lipid peroxidation. Taurodeoxycholate markedly increased the production of thiobarbituric acid-reactive substances, end products of lipid peroxidation, in isolated rat hepatocytes, whereas epimers of taurodeoxycholate did not. Deoxycholic acid inhibited mitochondrial NADH dehydrogenase and NADH: ferricytochrome c oxidoreductase activities, leading to free radical generation, whereas epimers of deoxycholic acid had no effect on mitochondrial enzymes. These findings suggested that hydrophobic bile acids cause lipid peroxidation by impairment of mitochondrial function, leading to the generation of free radicals; and epimerization of α-hydroxyl groups in the steroid nucleus to β-hydroxyl groups results in a decrease of the toxic effects of deoxycholic acid on lipid peroxidation.View full abstractDownload PDF (1270K) -
Shohei Maekawa, Hiroshi Morii, Haruko Kumanogoh, Mayuko Sano, Yoshihis ...2001 Volume 129 Issue 5 Pages 691-697
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRaft is a mobile membrane subdomain enriched in sphingolipid and cholesterol and also various signaling molecules. Previous observation suggested that brain-derived rafts contain tubulin but that rafts of non-neural origin do not. We hypothesized that SCG10, one of the neuronal growth-associated proteins (nGAPs), might be a neuron-specific molecule that anchors tubulin to neuronal rafts, and we explored biochemically its subcellular localization, interaction with tubulin, and effects on microtubule dynamics. In postnatal rat brain extracts, SCG10 was recovered mostly in membrane-associated fractions, and at least half was included in the raft fraction that was also enriched in GAP-43 and NAP-22. SCG10-enriched brain rafts also contained tubulin, and chemical cross-linking experiments revealed that SCG10 was closely associated with tubulin. In addition, SCG10 was able to inhibit polymerization of tubulin. These results indicate that SCG10 is a component of neuronal rafts as are other nGAPs, and suggest that SCG10 may be involved in signaling events in membranes for cytoskeletal reorganization around neuronal rafts.View full abstractDownload PDF (1819K) -
Koh Shikata, Satoshi Ohta, Kouichi Yamada, Chikashi Obuse, Hiroshi Yos ...2001 Volume 129 Issue 5 Pages 699-708
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn essential eukaryotic DNA polymerase, DNA polymerase δ (pol δ), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol δ. Our studies show that p66 interacts tightly with other subunits of pol δ during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol δ could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol δ for PCNA. These results indicate that p66 is a functionally important subunit of human pol δ that stabilizes the pol δ complex and increases the affinity of pol δ for PCNA.View full abstractDownload PDF (3933K) -
Toshitsugu Yubisui, Fumi Takahashi, Takashige Takabayashi, Shigeki Fuj ...2001 Volume 129 Issue 5 Pages 709-716
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA for cytochrome b5 was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b5. The nucleotide sequence of the cDNA for the ascidian cytochrome b5 (Pmb5) consisted of about 1, 800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b5. A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anionexchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b5 with an asymmetric peak at 556nm and a shoulder at 560nm upon reduction with NADH and NADH-cytochrome b5 reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555nm, this spectrum being very similar to that of mammalian liver cytochrome b5. Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b5 in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.View full abstractDownload PDF (2493K) -
Hiroyuki Irie, Hiroshi Koshiba, Mamoru Koyama, Eiji Asakura, Hiroshi S ...2001 Volume 129 Issue 5 Pages 717-724
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnti-atherosclerotic effects of human macrophage colony-stimulating factor were investigated using rabbits fed a high cholesterol diet. Rabbits fed a diet containing 2% cholesterol for 59 days developed hyperlipidemia and atheromatous aortic plaques. They were then administered 80μg/kg/day of either macrophage colony-stimulating factor or human serum albumin, as a control, for the next 12 weeks. Compared with the control group, rabbits treated with macrophage colony-stimulating factor had significantly fewer plaques on the inner surface of the thoracic and abdominal aortae, and half the sectional area of thickened intima in the aortic arch, as well as in the thoracic and abdominal aortae. Macrophage colony-stimulating factor also decreased the cholesterol content of the atherosclerotic lesions. Serobiochemical analyses revealed that macrophage colony-stimulating factor increased the levels of high density lipoprotein-cholesterol significantly, without influencing other lipid parameters such as the level of low density lipoproteins. The effects of macrophage colony-stimulating factor were evident until the fourth week of drug injection, at which time anti-human macrophage colonystimulating factor antibodies were clearly induced in the serum. These results indicate that exogenously administered macrophage colony-stimulating factor suppresses atherosclerotic lesions induced by a high cholesterol diet by activating lipid metabolism in vivo.View full abstractDownload PDF (2271K) -
Manami Kawabata, Maki Onda, Tomoyoshi Mita2001 Volume 129 Issue 5 Pages 725-732
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of aggregation of amphotericin B (AmB), as well as the complex formation of AmB with cholesterol or ergosterol, was investigated in micelles and vesicles. AmB in lysophosphatidylcholine (LPC) micelles adopted a more favorable monomeric form than that in other drug formulations. At an LPC/AmB ratio of 200, AmB existed only in monomeric form. Such monomeric behavior is likely dependent upon the fluidity and size of the micelles. In LPC micelles composed of 90% monomeric AmB, AmB-ergosterol complex formation occurred with an increase in the sterol concentration, but the complex formation of AmB-cholesterol was slight. On the other hand, in LPC micelles composed of 40% monomeric AmB, the complex formation of AmB-cholesterol as well as AmB-ergosterol was extensive. These results suggest that the complex formation of AmB with both sterols is highly dependent upon the aggregated state of AmB. In addition, using monolayers, mixtures of AmB/LPC/ergosterol were became more stable with rising temperature, while the stability of mixtures of AmB/LPC/cholesterol remained unchanged, implying that complex formation of AmB with cholesterol is different from that of AmB with ergosterol.View full abstractDownload PDF (1255K) -
Yoshihiro Ito, Guoping Chen, Yukio Imanishi, Takaya Morooka, Eisuke Ni ...2001 Volume 129 Issue 5 Pages 733-737
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC 12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.View full abstractDownload PDF (1637K) -
Tetsushi Amano, Tadahide Furuno, Naohide Hirashima, Nobuyuki Ohyama, M ...2001 Volume 129 Issue 5 Pages 739-744
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5μm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1±0.02μm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3min if the diameter of the cells is 20μm.View full abstractDownload PDF (2285K) -
Atsuhiko Ishida, Yasushi Shigeri, Yoshiro Tatsu, Yasuko Endo, Isamu Ka ...2001 Volume 129 Issue 5 Pages 745-753
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca2+/cahnodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMK-Pase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMK-Pase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the Vmax values.View full abstractDownload PDF (2354K) -
Norimasa Nakamura, David A. Hart, Cyril B. Frank, Linda L. Marchuk, Ni ...2001 Volume 129 Issue 5 Pages 755-759
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.View full abstractDownload PDF (1906K) -
Yuko Nitahara, Kae Kishimoto, Yoshiyasu Yabusaki, Osamu Gotoh, Yuzo Yo ...2001 Volume 129 Issue 5 Pages 761-768
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, 1.372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the “conserved threonine” of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51_ Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.View full abstractDownload PDF (2232K) -
Kazuhiro Karaya, Tatsuhito Shimizu, Akira Taketo2001 Volume 129 Issue 5 Pages 769-775
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptolysin S (SLS) is a serum-extractable and oxygen-stable hemolysin produced by Group A Streptococcus. A SLS-deficient mutant in which transposon Tn 916 was inserted in a locus distinct from the sag gene cluster [Nizet et al. (2000) Infect. Immun. 68, 4245-4254] was obtained by filter mating of the transposon-harbouring Enterococcus faecalis strain and Streptococcus pyogenes BL (T). This mutant, N22, had completely lost the hemolytic activity, in consequence of insertion of a single Tn 916 into a hitherto-unknown lantibiotic gene cluster composed of 10 open reading frames. The arrangement and sequence of this lantibiotic gene cluster were similar to those of nisin and subtilin, and so we designated this new lantibiotic as streptin. The bactericidal activity of streptin was abolished on treatment with trypsin or proteinase K. The different host range and nucleotide sequence clearly distinguished streptin from streptococcins. Streptin was not hemolytic and its bacteriocin activity was independent of carrier oligonucleotides effective for SLS. The fact that N22 also lost the anti-bacterial activity against indicator streptococci reveals that the factor (s) required for lantibiotic formation plays an important role in SLS formation as well.View full abstractDownload PDF (1378K) -
Junichi Miyazaki, Shuichi Nakaya, Toshiharu Suzuki, Masatada Tamakoshi ...2001 Volume 129 Issue 5 Pages 777-782
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAncestral amino acid residues were inferred for 3-isopropylmalate dehydrogenase (IPMDH), and were introduced into the enzyme of an extreme thermophile, Sulfolobus sp. strain 7. The thermostability of the mutant enzymes was compared with that of the wild type enzyme. At least five of the seven mutants tested showed higher thermal stability than the wild type IPMDH. The results are compatible with the hyperthermophilic universal ancestor hypothesis. The results also provide a new method for designing thermostable enzymes. The method only relies on the first dimensional structures of homologous enzymes that can be obtained from genetic databases.View full abstractDownload PDF (2020K) -
Shin Ono, Makiko Umezaki, Nagisa Tojo, Shuko Hashimoto, Hiroko Taniyam ...2001 Volume 129 Issue 5 Pages 783-790
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTendamistat is a strong inhibitory protein of porcine pancreatic α-amylase (PPA) with a Ki value of 0.2nM. To develop potent α-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (Ki=0.27μM) exhibited the strongest inhibitory activity (Ki=0.27-1.41μM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (Ki=0.28-1.00μM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (Ki=0.28μM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the β-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.View full abstractDownload PDF (1082K) -
Jun-ichi Aikawa, Young-Nam Park, Masakazu Sugiyama, Makoto Nishiyama, ...2001 Volume 129 Issue 5 Pages 791-794
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSite-directed mutagenesis was carried out to investigate the functional roles of amino acid residues of Rhizomucor pusillus pepsin (RMPP) in substrate-binding and catalysis. Mutations of two amino acid residues, E13 in the S3 subsite and N219 in the S3/S4 subsites, caused marked changes in kinetic parameters for two substrate peptides with different sequences. Further site-directed mutagenesis at E13 suggested that E13 plays a critical role in forming the correct hydrogen bond network around the active center. In the crystal structure of Rhizomucor miehei pepsin (RMMP), which is an aspartic proteinase produced by Rhizomucor miehei and shows 81% amino acid identity to RMPP, the OE atom of N219 forms a hydrogen bond with the N-H of isovaline in pepstatin A, a statine-type inhibitor, at the P3 position, suggesting that the loss of the hydrogen bond causes an unfavorable arrangement of the P3 residue. Among the mutants constructed, the E13A mutant showed a 5-fold increase in the ratio of clotting versus proteolytic activity without significant loss of clotting activity. This mutant may present a promising candidate for a useful milk coagulant.View full abstractDownload PDF (1095K) -
Shuji Miyagawa, Mako Yamada, Katsuyoshi Matsunami, Yukie Koresawa, Mas ...2001 Volume 129 Issue 5 Pages 795-801
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe human DAF (CD55) gene was chosen as a representative molecule in a xenotransplantation study. The gene was synthesized in order to adapt its codons to those which are more frequent in mammals, especially pigs, and the expression levels were then examined in Chinese hamster ovarian (CHO) cells, swine endothelial cell (SEC) and transgenie mice. A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAY (sDAF), compared with the wild-type DAF (wtDAF) and delta-SCR1 wild-type DAF (Δ1wtDAF). Consistent with the in vitro data, the expression of DAF in mice that carry sDAF was higher than Δ1wtDAF in many organs, especially the pancreas. The sDAF showed a high level of expression in SEC and transgenic mice, suggesting that it will be useful in the development of transgenie pigs with high levels of expression.View full abstractDownload PDF (1971K) -
Noriko Fujiwara, Tsuneko Fujii, Junichi Fujii, Naoyuki Taniguchi2001 Volume 129 Issue 5 Pages 803-812
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMammalian thioredoxin reductase [EC 1. 6. 4. 5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-G1y). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculoviruslinsect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Seccontaining thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5, 5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.View full abstractDownload PDF (1513K) -
Heat Shock Factor 2 Is Involved in the Upregulation of αB-Crystallin by High Extracellular PotassiumChiharu Sadamitsu, Toko Nagano, Yasuyuki Fukumaki, Akiko Iwakiz2001 Volume 129 Issue 5 Pages 813-820
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSαB-Crystallin, a member of the small heat shock protein (HSP) family, accumulates in reactive astrocytes in a variety of pathological conditions. We previously reported the upregulation of αB-crystallin in response to high extracellular potassium concentration. In the present study, we investigated the regulatory mechanism of aB-crystallin expression by KCL When human glioma U-251MG cells were exposed to continuous KCl treatment, induction of αB-crystallin mRNA was observed after 8h and persisted for a few days. Functional promoter analysis using deletion and mutation constructs revealed that the proximal heat shock element (HSE-P), which contributes to heat shock induction in HeLa cells, is essential for transcriptional activation of the eB-crystallin gene by KCl in U-251MG cells. Gel mobility shift and antibody supershift assays showed that KCl induces the HSE-binding activity of heat shock factor (HSF) 2, while heat shock induces that of HSF1. This is the first demonstration that HSF2 can be activated by KCl and is involved in the upregulation of αB-crystallin gene expression in glial cells.View full abstractDownload PDF (2595K) -
Jian Li, Sen Zhang, Chih-chen Wang2001 Volume 129 Issue 5 Pages 821-826
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReduced apo-α-lactalbumin (r-LA) in the pre-molten globule state is soluble in neutral and reduced buffer at 25°C but becomes aggregated when aggregates of various proteins are added. However, protein aggregates do not induce the aggregation of apo-α-lactalbumin in the molten globule state. The presence of the molecular chaperone protein disulfide isomerase or the “chemical chaperone” polyethyleneglycol inhibits the induced aggregation. Native proteins, aggregation-free folding intermediates, and soluble aggregates do not induce the aggregation. The interaction between r-LA and protein aggregates is hydrophobic in nature. These findings suggest that pre-molten globule state of LA is the target not only for chaperones but also for protein aggregates.View full abstractDownload PDF (1030K) -
Reona Takabatake, Abu-Baker M. Siddique, Hiroshi Kouchi, Katsura Izui, ...2001 Volume 129 Issue 5 Pages 827-833
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mitochondrial phosphate transporter of Saccharomyces cerevisiae, encoded by MIR1 (YJR077C) gene, shows divergence among the transporters in various eukaryotes. We have characterized another gene, YER053C, that appeared to encode an orthologous mitochondrial phosphate transporter of yeast. The predicted amino acid sequence of the YER053C protein is much more similar to that of mitochondrial phosphate transporters of other species than that of MIR1. RNA gel blot analysis indicated that, like the MIR1 promoter, the YER053C promoter is functional and that its activity varies according to aeration. An MIR1 gene null mutant did not grow on glycerol medium, whereas a YER-053C null mutant grew well on the medium, suggesting that the YER053C gene is not essential for the mitochondrial function. YER053C also did not support the growth of the MIR1null mutant on glycerol. The MIR1 and YER053C proteins were expressed in Escherichia coli and then reconstituted into liposomes. Unlike the proteoliposomes of MIR1, those of YER053C did not exhibit significant phosphate transport activity. Unexpectedly, it was shown that YER053C is localized in vacuoles, not mitochondria, by immunological electron microscopy. These results suggest that, during evolution, yeast lost the function and/or mitochondrial targeting of YER053C and then recruited an atypical MIRI as the only transporter.View full abstractDownload PDF (1969K) -
Brian A. Perrino, Brett A. Martin2001 Volume 129 Issue 5 Pages 835-841
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been proposed that N-terminal myristoylation of calcineurin B is necessary for the membrane association of calcineurin. We tested the effects of Ca2+ and myristoylation on the binding of calcineurin B alone or heterodimeric calcineurin to phosphatidylserine or phosphatidylcholine vesicles. In the presence of excess phosphatidylserine, 50-60% of total calcineurin associated with phosphatidylserine in a Ca2+-sensitive manner. Calcineurin did not associate with phosphatidylcholine. Calcineurin containing both the α and β catalytic subunit isoforms bound to phosphatidylserine. Cahnodulin interfered with the association of calcineurin with phosphatidylserine. In the presence of Ca2+, myristoylated calcineurin B alone did not bind to phosphatidylcholine but did bind to phosphatidylserine, although to a lesser extent than the calcineurin heterodimer. Non-myristoylated calcineurin B alone, or calcineurin containing non-myristoylated calcineurin B did not associate with phosphatidylserine in the presence of Ca2+. These results indicate: (i) Both isoforms of calcineurin bind to phosphatidylserine. (ii) A phospholipid binding site is located on the calcineurin B subunit. (iii) Calcineurin B myristoylation is required for the Ca2+-sensitive binding of calcineurin to phosphatidylserine vesicles in vitro.View full abstractDownload PDF (3268K) -
Brett A. Martin, Brian C. Oxhorn, Charles R. Rossow, Brian A. Perrino2001 Volume 129 Issue 5 Pages 843-849
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions between phospholipid membranes and the acyl chain and specific amino acid residues of myristoylated proteins are necessary for membrane association. In the present study we tested the effects of mutations of calcineurin B subunit amino acid residues K20K21, K24R25, K27K28 to Glu on the interactions between calcineurin and phosphatidylserine vesicles. Calcineurin-phosphatidylserine interactions were measured using binding assays and assays of phosphatidylserine-stimulated calcineurin phosphatase activity. The reverse-charge calcineurin B subunit mutant had a slower mobility in SDS-PAGE relative to wild-type calcineurin B. In addition, the myristoylated calcineurin B reverse-charge mutant had a slower mobility in SDS-PAGE compared to the non-myristoylated form, in contrast to the faster mobility of myristoylated wild-type calcineurin B relative to non-myristoylated calcineurin B. The reverse-charge mutations had no apparent effect on N-terminal myristoylation, Ca2+-binding, or calcineurin heterodimer formation and stimulation of Ca2+/calmodulin-dependent phosphatase activity. However, in contrast to the results obtained using native calcineurin, phosphatidylserine vesicles did not bind to or activate the phosphatase activity of calcineurin containing the calcineurin B reverse-charge mutant. These results indicate that calcineurin B contains an amino terminal basic residue cluster that is involved in the binding of calcineurin to acidic phospholipids.View full abstractDownload PDF (3451K)
- |<
- <
- 1
- >
- >|