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Volume 117, Issue 1
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Koscak Maruyama1995 Volume 117 Issue 1 Pages 1-6
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhy were the two classical papers by A. F. Huxley and R. Niedergerke and by H. E. Huxley and J. Hanson on the sliding filament concept in muscle contraction published in the same issue (May 22, 1954) of Nature This historical survey reveals the background of the two groups' monumental work.View full abstractDownload PDF (1838K) -
Nozomi Yamaguchi, Kunihiko Koyama, Yoshiro Yamamura, Akira Okui, Eiich ...1995 Volume 117 Issue 1 Pages 7-10
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSixty-four kinds of cell lines were examined as to their ability to degrade glucagon using conditioned-media obtained from their protein-free cultures. Two human tumor cell lines were shown to produce this activity, and the cell line, HPC-YO, established from a human pancreatic carcinoma was shown to produce the highest level of activity. The glucagondegrading enzyme (GDE) was purified from HPC-YO conditioned-medium by a combination of ion-exchange, gel filtration, and hydroxylapatite column chromatographies. The purified GDE also degraded vasoactive intestinal polypeptide (VIP) and secretin, however, it did not cleave EGF, gastrin, insulin, somatostatin, substance P, neurotensin, or growth hormone. The molecular weight of GDE is 83, 000, as determined on SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of GDE was blocked, and the five partial amino acid sequences obtained on lysyl-endopeptidase digestion were determined to be N-L-T-E-E-Y-D-V-S-D-G-E-I-E-L-L-Y-E-K, V-E-T-Y-Y-D-L-L-F-E-K, L-Y-W-F-L-D-E-A-K, S-N-S-T-S-Y-V-K, and Y-Y-A-S-T-S-Y-D-D-T-Y-K. The same or homologous amino acid sequences have not been found in known proteins, demonstrating that GDE is a novel peptidase that degrades the secretin family: glucagon, VIP, and secretin.View full abstractDownload PDF (453K) -
Hidekazu Ohizumi, Yutaka Masuda, Shigeo Nakajo, Itaru Sakai, Shigemits ...1995 Volume 117 Issue 1 Pages 11-13
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe screened various isoprenoids to find inducers of apoptosis for human leukemia HL-60 cells, and found that GGO (geranylgeraniol) had the most potent apoptosis-inducing activity, as judged from DNA fragmentation in HL-60 cells. The apoptosis-inducing activity of GGO is concentration- and time-dependent. DNA synthesis by HL-60 cells was selectively inhibited on treatment with GGO. Besides HL-60 cells, apoptosis was induced by GGO in various tumor cell lines, including human myeloid multipotential leukemia K562, lymphoblastic leukemia Molt3, and colon adenocarcinoma COLO320 DM.View full abstractDownload PDF (2234K) -
Ken-ichi Saguchi, Takashi Tobe, Ken Hashimoto, Yoshihiro Sano, Yasuko ...1995 Volume 117 Issue 1 Pages 14-18
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cDNA encoding inter-α-trypsin inhibitor family heavy chain-related protein (IHRP) was cloned from human liver cDNA libraries. Oligonucleotide primers of human liver cDNA for PCR were constructed from internal amino acid sequences obtained with proteolytic fragments of IHRP. The amplified cDNA served as a hybridization probe for the screening of human liver cDNA libraries. The cDNA of 2, 977 by contained an entire reading frame coding 930 amino acids. The N-terminal 28 residues corresponded to a signal peptide for secretion. The N-terminal 600 residues of the mature form exhibited considerable homology to those of ITI heavy chains, while the C-terminal 300 residues showed no homology with the heavy chains and low homology with ATP-dependent proteases. IHRP was readily cleaved into 85- and 35-kDa fragments when plasma was incubated at 37°C. The cleaved site, Arg-Arg-Leu, was within a proline-rich region. Northern blot analysis of poly(A) RNAs from various human tissues only showed hybridization to liver RNA.View full abstractDownload PDF (2119K) -
In Kwon Chung, Stephen M. Soisson, Mark T. Muller1995 Volume 117 Issue 1 Pages 19-22
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe report that a host nuclear protein of approximately 100 kDa binds to the tandemly reiterated DR2 sequence of herpes simplex virus type 1 (HSV-1). The DR2 sequence is a repeated component in the “a” sequence, which defines the signals for cleavage and encapsidation of viral DNA; the “a” sequence also contains the promoter regulatory signals for the gene encoding the viral neurovirulence factor, ICP34.5. Characterization of the host binding protein by means of gel shifts and DNase I footprinting revealed this protein is the eukaryotic transcription factor, Sp1. Furthermore, as judged from the sequence homology, the DR2 region contains clustered matches to the consensus binding site for Sp1. Comparison of the host factor and purified Sp1 (by means of gel shifts and footprinting) confirmed these findings. Since clustered DNA recognition elements represent unusually high affinity binding sites, these repeated Sp1 motifs proximal to the ICP34.5 gene suggest that this region may be a major Sp1 binding site in the viral genome.View full abstractDownload PDF (2898K) -
Mikio Shimizu1995 Volume 117 Issue 1 Pages 23-26
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA series of C4N hairpin RNAs bearing anticodon nucleotides at the 5' ends and a discriminator base and the sequence CCA at the 3' ends was constructed by an in vitro transcription system using T7 RNA polymerase. These RNAs were aminoacylated specifically with their cognate amino acids by reaction with aminoacyl-adenylates in the presence of a dipeptide, valyl-aspartic acid, suggesting that such hairpin RNAs are able to play the role of the present-day tRNA and that valyl-aspartic acid can perform the function of the present-day aminoacyl-tRNA synthetase as a catalyst in the aminoacylation reaction. These results should provide a useful clue to elucidating the origin of the genetic code.View full abstractDownload PDF (3095K) -
Kazuko Ohgi, Masanori Iwama, Kuriko Tada, Ritsue Takizawa, Masachika I ...1995 Volume 117 Issue 1 Pages 27-33
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to elucidate on the mechanism of action of RNase Rh from Rhizopus niveus, we investigated the role of Lys108, which is conserved among the RNase T2 family RNases except for two cases. The RNase activities of Lys108 mutant RNases, RNase RNAP K108R and K108L, are about 33.5 and 3.1% of that of the wild type enzyme, respectively. The relative rates of cleavage of dinucleoside phosphates by these two mutant enzymes were comparable to those with RNA as a substrate. The kinetic parameters of RNases RNAP K108R and K108L towards XpGs (where X is one of A, G, U, and C) were measured. The data indicated that the Km values of the two mutant enzymes are similar to those of the wild-type enzyme. The rates of release of the four nucleotides from RNA by digestion with the mutant enzymes were in the order A>G>U>C, which is qualitatively the same as that of the wild-type enzyme. From these data, we concluded that the Lys108 residue participates in the catalytic process, but not in the binding, and the positive charge of Lys108 is indispensable for the catalytic process, that is, the positive charge of Lys108 may stabilize the pentacoordinated intermediate in the transition state as proposed in the case of Lys41 in RNase A, or may polarize the phosphate moiety of the substrate.View full abstractDownload PDF (2745K) -
Tetsuro Yoshimura, Masayuki Shono, Katsuyuki Imai, Keelung Hong1995 Volume 117 Issue 1 Pages 34-41
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndocytosis and the intracellular fate of liposomes in single mouse peritoneal macrophages were examined kinetically by fluorescence microphotometry. Liposomes labeled with N- (7-nitro-2, 1, 3-benzoxadiazol-4-yl) phosphatidylethanolamine or containing 8-amino-naphthalene-1, 3, 6-trisulfonate were promptly incorporated into macrophages on incubation at 37°C, but fluorescence increase caused by hydrolysis of 4-methylumbelliferyl-β-D-glucoside encapsulated in the liposomes was observed after 30min of incubation. The fluorescences of calcein and 8-hydroxy-1, 3, 6-pyrenetrisulfonate (HPTS) in liposomes, which were respectively quenched statically due to high concentration and dynamically by a co-entrapped fluorescence quencher, p-xylene-bis-pyridinium bromide, also increased from 30min after the start of liposome incorporation, indicating that macrophages require this period for intracellular delivery of liposomes from the cell surface to lysosomes. Measurement of the intraendosomal pH change in a single macrophage at 37°C with liposomes containing a pH-sensitive fluorescent marker, HPTS, showed that the pH value decreased continuously to a constant value of 5.5 in 30-40min after endocytosis, and this decrease was reversed on addition of NH4Cl, suggesting that acidification of endosomes is not a stepwise reaction and is coupled with delivery of liposomes. These fluorescence microphotometric systems using liposomes containing different fluorescent dyes should be useful for kinetic analyses of the endocytosis and intracellular fate of liposomes in various phagocytes.View full abstractDownload PDF (3719K) -
Yasuhisa Kono1995 Volume 117 Issue 1 Pages 42-46
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEscherichia coli was killed by catalase in dose-, time-, and pH-dependent manners. Dialyzed catalase had bactericidal activity, but enzyme which had been heat-denatured or inactivated by pretreatment with 3-aminotriazole plus hydrogen peroxide did not. Cytochrome c and hemoglobin also had bactericidal activity. Thiobarbituric acid-reactive substances were detected in commercial hemoproteins except for horseradish peroxidase and the relationship between the contents of these substances and bactericidal activity was demonstrated. Without the addition of hydroperoxide, hemoproteins except for horseradish peroxidase initiated the oxidation of 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and catalase caused the peroxidation of linoleic acid. The pH patterns of bactericidal activity, lipid peroxidation, and the oxidation of ABTS were similar. The results indicate that hemoprotein preparations are contaminated with lipid hydroperoxides and it is the decomposition of these contaminants catalyzed by the hemoprotein into alkoxyl/peroxyl radicals that causes bacterial killing.View full abstractDownload PDF (570K) -
Masashi Saito, Konami Kita, Kunio Sekiya, Saburo Omata, Tsuneyoshi Hor ...1995 Volume 117 Issue 1 Pages 47-53
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 144 kDa protein was purified from the WGA-Sepharose bound fraction of a rat liver nuclear envelope salt-extract by hydroxyapatite HPLC (HAP HPLC). Two other, 120 and 86 kDa, proteins were also partially purified from the fraction by a combination of DEAE- and HAP-HPLCs. It was suggested that the 144, 120, and 86 kDa proteins bear G1cNAc residues, and are nucleoporins, because they were purified from nuclear envelopes, reacted with WGA-HRP, and cross-reacted with an antibody against p62 nucleoporin complexes. The sedimentation coefficients and Stokes' radii of these G1cNAc-bearing proteins were determined by glycerol density gradient centrifugation and gel filtration in the presence of 500mM NaCl. The molecular masses calculated from these values suggested that these three proteins each exist as a monomer under the conditions employed. The axial ratios of the purified 144, 120, and 86 kDa G1cNAc-proteins were estimated to be 35, 31, and 31, respectively. These values suggested that they are rod-shaped molecules. The axial ratio of a purified nucleoporin-complex consisting of 62, 60, and 54 kDa components bearing GlcNAc was shown to be 20. This nucleoporin complex seems to be a rod-shaped complex. From these results, a rod shape is proposed to be a common characteristic of G1cNAc-proteins in nuclear envelopes.View full abstractDownload PDF (4209K) -
Eun-Ok Lee, Jong-Duk Kim1995 Volume 117 Issue 1 Pages 54-58
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction with fungal cells of dioleoylphosphatidylethanolamine liposomes coated with palmitoyl lysozyme was investigated using lysozyme as a stabilizer for an unstable bilayer as well as a recognizer for a specific target cell. Lysozyme, which interacts with chitin in the fungal cell wall, lyses chitin and kills the cells, was acylated with N-hydroxysuccinimide ester of palmitic acid (NHSP) and incorporated into the lipid bilayer. Lysozyme was optimally modified when the ratio of NHSP to lysozyme was 15 at pH 8.0. Modification of lysozyme was detected by SDS-PAGE, and its molecular weight was about 1, 500 greater than that of the intact lysozyme at the optimal ratio of NHSP to lysozyme. The activity of palmitoyl lysozyme toward glycolchitin was reduced to 35% of that of intact lysozyme. Both lysozyme and palmitoyl lysozyme had antifungal activities, but palmitoyl lysozyme was more effective than intact lysozyme against Candida albicans. The minimal molar ratio of palmitoyl lysozyme to phosphatidylethanolamine required to form stable liposomes was 2.4×10-4, and the optimal ratio was about 2.4×10-3. The percentage survival of cells treated with the inserted palmitoyl lysozyme was lower than that of cells treated with free palmitoyl lysozyme. These findings suggest that palmitoyl lysozyme-incorporated liposomal membrane is more effectively adsorbed by Candida albicans than free palmitoyl lysozyme is.View full abstractDownload PDF (1374K) -
Akira Noguchi, Chege J. Mukuria, Eiko Suzuki, Masaharu Naiki1995 Volume 117 Issue 1 Pages 59-62
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant human erythropoietin (EPO) produced by Chinese hamster ovary cells and distributed by two different pharmaceutical companies were confirmed to contain about 1% N-glycolylneuraminic acid (Neu5Gc) in total sialic acid content. Since chickens, like humans, do not synthesize Neu5Gc, they were used to determine the immunogenicity of Neu5Gc epitope in EPO. Chickens immunized with EPO did not produce significant titer of antibody that was specific to GM3 (Neu5Gc) as compared to antibody titers produced in chickens immunized with fetuin containing 7% Neu5Gc or GM3 (Neu5Gc) containing 100% Neu5Gc. Results obtained by an ELISA inhibition test showed that EPO, compared to GM3 (Neu5Gc), reacted almost one thousand times less strongly with a human Hanganutziu-Deicher (HD) antibody. This study implies that an increase of Neu5Gc content in a molecule enhances its HD antigenicity. The response to Neu5Gc in patients receiving therapeutic injections of EPO is currently under investigation.View full abstractDownload PDF (470K) -
Naohiro Yoshigi, Hirohisa Sahara, Shohei Koshino1995 Volume 117 Issue 1 Pages 63-67
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the role of the C-terminal region of barley β-amylase, plasmid Δ54 was constructed with an expression vector (pBETA92) of barley β-amylase by site-directed mutagenesis. Escherichia coli JM109 harboring plasmid Δ54 was expected to express Δ54 β-amylase in which 54 amino acid residues were deleted from the C-terminus. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. Δ54 β-amylase gave a single activity band on isoelectric focusing (pI 6.85). Δ54 β-amylase was purified from the cells by consecutive α-cyclodextrin/Sepharose 6B column chromatography. A comparison of the properties of the mutant enzyme with those of the original recombinant β-amylase [Biosci. Biotech. Biochem. (1994) 58, 1080-1086] revealed two major differences. First, the original recombinant β-amylase showed heterogeneity on isoelectric focusing, but Δ54 β-amylase gave a single main band of protein (pI 6.85). Therefore, the isoelectrophoretic heterogeneity of the original recombinant β-amylase was apparently due to its C-terminal region. Secondly, Δ54 β-amylase lacked thermostability. Therefore, it was concluded that the C-terminal region was significantly involved in the thermostability of β-amylase.View full abstractDownload PDF (1365K) -
Takanori Katsube, Yasuyuki Fukumaki1995 Volume 117 Issue 1 Pages 68-76
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHereditary persistence of fetal hemoglobin (HPFH) is a condition manifested by continued expression of the γ-globin gene in adult life. The four types of HPFH mutations (-117 G-A, -114 C-T, -114 C-G, and 13 by del) clustered in the vicinity of the distal CCAAT box of the γ-globin gene suggest the relevance of this region to the fetal-to-adult hemoglobin switching. Functional analysis in erythroid cell lines showed that these HPFH mutations reduced the activity of the γ-globin promoter in K562 expressing embryonic and fetal globin chains, but not in KU812 expressing fetal and adult globin chains. These results suggest that the distal CCAAT box region has different functions in the fetus and the adult. In vitro, binding of NFE3 to the distal CCAAT box was commonly reduced by these HPFH mutations, suggesting that the binding of NFE3 to the distal CCAAT box may repress the γ-globin gene in adults. Overlap of the binding sites of NFE3 and CP1, a potential activator, indicates the regulation of the γ-globin gene by the competition between these factors. In addition, an unknown factor interacting with the -114 C-T HPFH mutant CCAAT box may be involved in elevation of the γ-globin expression in an individual with the -114 C-T mutation.View full abstractDownload PDF (6557K) -
Yasuhito Terui, Yusuke Furukawa, Kumi Sakoe, Masatsugu Ohta, Masaki Sa ...1995 Volume 117 Issue 1 Pages 77-84
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen human promyelocytic leukemia cell line HL-60 was treated with various differ-entiation-inducers, apoptosis always occurred after the full appearance of differentiation-related phenotypes. However, the two phenomena could be dissociated when HL-60 cells were treated with PDBu. When HL-60 cells were cultured with PDBu for more than 36 h, apoptosis was induced following differentiation. Apoptosis was not, however, observed when PDBu was removed within 24 h, even though induction of differentiation-related phenotypes, such as NBT-reducing ability and surface marker expression, was the same as that in the control. Northern blot analysis revealed that bcl-2 mRNA was rapidly downregulated within 6 h of the treatment with PDBu. The amount of bcl-2 mRNA recovered to that of undifferentiated HL-60 cells when PDBu was washed out within 24 h. In contrast, the recovery of bcl-2 was incomplete when the cells were treated with PDBu for more than 36 h, suggesting that bcl-2 is also a critical regulator of the cell fate during myeloid differentiation. This hypothesis was confirmed by experiments using antisense oligonucleotides, i.e., blocking the recovery of bcl-2 mRNA by antisense oligonucleotides could result in the induction of apoptosis in HL-60 cells from which PDBu was removed within 24 h. Moreover, overexpression of BCL-2 in HL-60 cells could block apoptosis during differentiation without any significant effect on differentiation itself. These results strongly suggest that apoptosis is not a simple consequence of differentiation-induction, and that apoptosis and differentiation are regulated independently in myeloid cells.View full abstractDownload PDF (8331K) -
Isamu Kameshita, Hitoshi Fujisawa1995 Volume 117 Issue 1 Pages 85-90
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur earlier finding [Kameshita, I. and Fujisawa, H. (1993) J. Biochem. 113, 583-590] that calmodulin-dependent protein kinase IV (CaM-kinase IV) from rat cerebral cortex is markedly activated through autophosphorylation was reexamined in the light of a more recent study [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170] suggesting the involvement of Ca2+/calmodulin-dependent CaM-kinase IV kinase in the activation of CaM-kinase IV in rat brain. Further purification of the previous CaM-kinase IV preparation by HPLC on a DEAE-NPR column abolished its marked activation on incubation under Ca2+/calmodulin-dependent phosphorylation conditions, and the marked activation was restored by the addition of the fraction separated on a DEAE-NPR column, suggesting that CaM-kinase IV was separated from CaM-kinase IV kinase involved in its activation by HPLC on DEAE-NPR. The HPLC-purified CaM-kinase IV still underwent a slow autophosphorylation, which was an intramolecular reaction. Some kinetic properties of CaM-kinase IV before and after autophosphorylation were examined for comparison. In addition, the purification by HPLC on DEAE-NPR revealed that a novel Ca2+-dependent calmodulinbinding protein, designated as CaMBP64, is present as a major contaminant in the previous purified CaM-kinase IV preparation.View full abstractDownload PDF (3001K) -
Akira Kobayashi, Kazuhiro Sogawa, Hiroaki Imataka, Yoshiaki Fujii-Kuri ...1995 Volume 117 Issue 1 Pages 91-95
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBTEB is a small GC box-binding protein containing three contiguous zinc finger structures in the C-terminal region of the molecule, and activates or represses the transcription of genes with the GC box sequence in the promoter, depending on the repetitiveness of the GC box sequence [Imataka et al. (1992) EMBO J. 11, 3663-3671]. We have analyzed functional domains of BTEB in a transient expression system using Y-1 cells (a mouse adrenal cortex cell line). BTEB contained two regions responsible for the transcriptional activation. These regions showed a sequence similarity to each other and appeared to enhance the transcription independently. The two sequences were rich in hydrophobic amino acids and showed no similarity to the sequences of transactivation domains so far elucidated. The DNA-binding properties of BTEB were also analyzed by means of the gel mobility shift assay. Three contiguous zinc finger motifs were needed for the binding activity. Furthermore, it was revealed that a short basic region immediately N-terminal to the zinc finger motifs was required for the DNA binding.View full abstractDownload PDF (3034K) -
Katsumi Tsukazaki, Hideki Nikami, Yasutake Shimizu, Teruo Kawada, Tosh ...1995 Volume 117 Issue 1 Pages 96-100
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen mammals are exposed to a cold environment for a long time, the capacity of nonshivering thermogenesis by brown adipose tissue (BAT) increases in association with the stimulation of synthesis of some specific proteins and tissue hyperplasia, which are totally dependent on sympathetic innervation to this tissue. To clarify the roles of the adrenergic mechanisms for the cold-induced protein synthesis and hyperplasia in BAT, in this study, the effects of chronic treatment with adrenergic agonists using an osmotic mini-pump were examined in rats. Continuous administration of noradrenaline or isoproterenol (β-agonist) for 10 days resulted in increased synthesis of the mitochondrial uncoupling protein and an isoform of glucose transporter (GLUT4), and tissue hyperplasia, in the same way as after cold exposure of the same duration. Phenylephrine (α-agonist) administration did not have any significant effect. Surgical sympathetic denervation completely abolished the effects of cold exposure, whereas it did not influence those of adrenergic agonists at all. These results indicate that the stimulative effects of cold exposure on protein synthesis and hyperplasia of BAT are attributable solely to the β-adrenergic action of noradrenaline secreted from the sympathetic nerves in this tissue.View full abstractDownload PDF (1540K) -
Takashi Hibino, Byung Hyun Lee, Tetsuko Takabe, Teruhiro Takabe1995 Volume 117 Issue 1 Pages 101-106
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the role of the copper-ligand Met92 in the structural and functional properties of silene plastocyanin (PC), Met92 was replaced with Gln, which is the purposed fourth copper-ligand in another blue copper protein, stellacyanin. By use of the recently developed expression system [Hibino et al. (1994) J. Biochem. 116, 826-832], the Met92Gln mutant of intermediate precursor plastocyanin was successfully expressed in Escherichia coli and accumulated in the periplasmic space as a mature protein. In contrast to the wild type, most of the Met92Gln mutant PC accumulated as an apoprotein. After purification, mutant apoprotein could incorporate copper ions, although less efficiently than the wild-type apoprotein. The absorption peak of Met92Gln mutant PC was blue-shifted from 597 nm in the wild type to 591 nm. The rhombic type EPR spectrum was obtained for the mutant in place of the axial spectrum in the wild type. Compared with that of the wild-type PC, the oxidation-reduction potentials of the Met92Gln mutant PC were lower by about 35 mV over the whole pH range examined. These results indicate that the Met92Gln mutant exhibited “stellacyanin-like” spectroscopic properties. Interestingly, the electron-transfer activities of the mutant PC with the physiological electron donor (cytochrome f) and acceptor (Photosystem I) were similar to that of the wild-type PC. Since Met92 is conserved in all the plastocyanins whose primary structures are known, we propose that the primarily function of Met as a copper-ligand is in the uptake of copper ions during folding rather than in electron-transfer activities.View full abstractDownload PDF (1736K) -
Kyoko Hiramatsu, Masayuki Sugimoto, Sachiko Kamei, Masato Hoshino, Ken ...1995 Volume 117 Issue 1 Pages 107-112
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn analytical system developed for fractionating free and monoacetylated polyamines [Hiramatsu, K. et al. (1994) J. Biochem. 115, 584-589] was proved useful also in detecting diacetylpolyamines, namely N1, N8-diacetylspermidine (diAcSpd) and N1, N12-diacetylspermine (diAcSpm). Detection limits were 0.9 and 0.6 pmol (S/N=5) for diAcSpd and diAcSpm, respectively. Analytical recovery and within-run variation were also satisfactory. Human urine samples were found to contain diAcSpd and diAcSpm. These polyamines were identified on the basis of the following observations: (i) their retention times were coincident with those of authentic samples; (ii) they were deacetylated to N8-acetylspermidine and monoacetyl- and free spermine, respectively, by acetylpolyamine amidohydrolase; and (iii) they were practically inert to direct oxidation by bacterial polyamine oxidase as were authentic samples. The amounts of eleven polyamine species including diAcSpd and diAcSpm in urine samples from 52 healthy persons were determined. Mean values for the major polyamine components were consistent with those reported by others. Although the amounts of diAcSpd and diAcSpm were very small, comprising only 1.4 and 0.46% of total polyamines, respectively, these two compounds were found to be always present in healthy human urine as regular constituents. Moreover, variation in their content among individuals was small, suggesting that excretion of these components in urine is strictly regulated.View full abstractDownload PDF (756K) -
Mamoru Sato, Yuji Ito, Mitsuo Harada, Hiroshi Kihara, Hirotsugu Tsurut ...1995 Volume 117 Issue 1 Pages 113-119
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ATP-hydrolyzing excitation state of the α3β3 complex of the ATP synthase from the thermophilic bacterium PS3 was investigated using time-resolved small-angle X-ray scattering with synchrotron radiation. The results showed the presence of the α3β3 complex at a steady state during ATP hydrolysis when the α3β3 hexamer reacted with Mg-ATP. The radius of gyration of the complex in the steady state was significantly larger than that of the Mg-AMP-PNP-hexamer complex, indicating a conformational change to an expanded structure during catalysis. This α3β3 complex dissociated into α1β1 heterodimers with apparent first-order reaction kinetics after all the ATPs were converted to ADPs. In contrast, when the α3β3 complex reacted with Mg-ADP, the complex dissociated into dimers with apparent first-order reaction kinetics without showing the steady state of the complex. The dimers, however, re-associated into the hexamer when Mg-ATP was added. The results were well-explained by a computer simulation based on non-linear chemical dynamics, in which a reaction mechanism that incorporates the dynamic structure of the hexamer in the steady state was considered.View full abstractDownload PDF (2102K) -
Kazuko Ôba, Seiko Ishikawa, Masumi Nishikawa, Hiroko Mizuno, Tom ...1995 Volume 117 Issue 1 Pages 120-124
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Galactono-γ-lactone dehydrogenase (L-galactono-γ-lactone:ferricytochrome c oxidoreductase [EC 1.3.2.3], GLDHase) which catalyzes the terminal step in the biosynthesis of L-ascorbic acid (AsA) has been purified from roots of sweet potato (Ipomoea batatas L., cv. Kintoki). Highly purified preparation of the GLDHase was obtained by three column chromatography steps with a recovery of ca. 1%, after solubilization from mitochondria in sweet potato roots. SDS-PAGE exhibited a single band at 56 kDa. In the native state, the apparent molecular mass of the enzyme was 56 kDa, based on a Sephadex G-100 gel filtration. The pI and optimum pH values were 5.8 and 7.9, respectively. The Km value for L-galactono-γ-lactone was 0.12mM. Substrate inhibition was obtained at concentrations greater than 4.2mM. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and acriflavine, and the inhibition of acriflavine was diminished by the addition of FAD or FMN. The only effective substrate for the GLDHase was L-galactono-γ-lactone.View full abstractDownload PDF (1607K) -
Enzyme Properties of Aplysia ADP-Ribosyl Cyclase: Comparison with NAD Glycohydrolase of CD38 AntigenKiyoshi Inageda, Katsunobu Takahashi, Ken-ichi Tokita, Hiroshi Nishina ...1995 Volume 117 Issue 1 Pages 125-131
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ecto-enzyme of NAD glycohydrolase (NADase) induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 antigen [Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898]. CD38 antigen has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase which generates cyclic adenosine diphosphoribose (cADPR) and nicotinamide (NA) from β-NAD+. On the basis of this sequence homology, we compared enzyme properties between CD38 NADase expressed as a fusion protein in Escherichia coli and ADP-ribosyl cyclase purified from the ovotestis of Aplysia kurodai. 1) β-NAD+ analogs, nicotinamide 1, N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide, did not serve as good substrates for the ADP-ribosyl cyclase, suggesting that the intact adenine ring of β-NAD+ was required for the cyclase-catalyzed reaction. On the other hand, CD38 NADase utilized the NAD analogs to form ADP-ribose and NA. 2) Kinetic analyses of the ADP-ribosyl cyclase reaction revealed that NA was first released from the substrate (β-NAD+)-enzyme complex, followed by the release of another product, cADPR, which was capable of interacting with the free enzyme. 3) The enzyme reaction catalyzed by the ADP-ribosyl cyclase was fully reversible; β-NAD+ could be formed from cADPR and NA with a velocity similar to that observed in the degradation of β-NAD+. However, CD38 NADase did not catalyze the reverse reaction to form β-NAD+ from ADP-ribose and NA. 4) The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol. These results indicated that enzyme reactions catalyzed by Aplysia ADP-ribosyl cyclase and CD38 NADase were quite different from each other in terms of their substrate specificities, reversible reactions, and susceptibilities to dithiothreitol, though both enzymes cleaved the N-glycoside bond of β-NAD+ resulting in the liberation of NA.View full abstractDownload PDF (2532K) -
Akinobu Hayashi, Tadashi Suzuki, Shingo Tajima1995 Volume 117 Issue 1 Pages 132-136
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEffects of retinoid derivatives (retinol, retinal, retinoic acid, and etretinate) on elastin expression and cell proliferation in chick embryonic vascular smooth muscle cells were compared. Retinoic acid and etretinate inhibited cell proliferation to a greater extent than retinol and retinal. Retinol showed no significant effect on cell proliferation or elastin synthesis. Retinoic acid exhibited the most pronounced stimulatory effect on elastin synthesis with a maximum stimulation of 2.8-fold at the concentration of 10-6 M for 24-48 h treatment. A comparable increase in elastin mRNA level was observed in the case of retinoic acid treatment. When the cells were serially passaged to the third subcultivation, elastin synthesis declined with increasing number of cell passages. Retinoic acid reversed this decline of elastin synthesis. The results indicate that retinoic acid was potentially active and retinol was inactive for both inhibition of cell proliferation and stimulation of elastin synthesis. The inhibitory activities of the four compounds for cell proliferation appear to parallel the stimulatory activities for elastin synthesis. The stimulation of elastin expression by retinoids may thus be closely related to the inhibition of cell proliferation.View full abstractDownload PDF (3799K) -
Kazuhito Inoguchi, Hidekatsu Yoshioka, Mohammed Khaleduzzaman, Yoshifu ...1995 Volume 117 Issue 1 Pages 137-146
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have isolated cDNAs and completed for the first time the primary structure for a novel collagenous chain that was partially characterized earlier and named α1 (Y) chain [Yoshioka, H. et al. (1992) Genomics 13, 884-886]. The size of the coding region was unexpectedly small compared with the length of the mRNA (>10 kb), owing to the presence of a long 3' untranslated region (>5 kb). The predicted polypeptide contained 1, 142 amino acid residues with a 23-residue signal peptide consisting of 5 collagenous domains of 70-224 residues in length, interspersed and flanked with 6 noncollagenous (NC) domains. The primary structure is distinct from those of the 32 known collagen α-chains of types I through XVIII. Therefore, we designate this newly discovered collagen chain the α1 chain of type XIX collagen. Sequence analysis suggested that this chain belongs to the recently discovered group of collagens known as FACITs (fibril associated collagens with inter-rupted triple-helices). Northern blotting analysis demonstrated hybridization of the cDNA to a large mRNA species (>10 kb) extracted from a rhabdomyosarcoma cell line (CCL 136). We also isolated numerous truncated cDNA clones of which the 3' parts were different from the “proto” type of the mRNA of >10-kb size. Sequence comparison between cDNAs and corresponding genomic DNA fragments indicated that unusual splicing events occurred through insufficient recognition at acceptor sites. Expression of the gene was extremely infrequent in the rhabdomyosarcoma cell line; it could be restricted to certain animal tissues both temporally and spatially during early development.View full abstractDownload PDF (4786K) -
Takeshi Sato, Koji Takio, Akira Kobata, Dale E. Greenwalt, Kiyoshi Fur ...1995 Volume 117 Issue 1 Pages 147-157
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous studies showed that the N-linked sugar chains of most bovine glycoproteins from milk fat globule membranes (MFGM) contain the GaINAcβ1→4GIcNAc group [Sato et al. (1993) J. Biochem. 114, 890-900]. Since expression of the disaccharide structure is influenced by peptide sequences near the glycosylation sites [Smith and Baenziger (1992) Proc. Natl. Acad. Sci. USA 89, 329-333], the site-specificity of the N-acetylgalactosaminylated sugar chains was investigated using bovine butyrophilin, a major MFGM glycoprotein with known primary structure. Two glycopeptide fragments which contained the N-linked sugar chains linked to either Asn-55 or Asn-215 residue were obtained by digestion of the protein with Achromobacter protease I. The sugar chains released from each glycopeptide by hydrazinolysis were reduced with NaB3H4. Structural analyses of the oligosaccharides by sequential exoglycosidase digestion and methylation analysis revealed that only complex-type sugar chains with the Ga1NAcβ1→4GIcNAc structure are included in Asn-55-linked oligosaccharides, while only novel hybrid-type sugar chains detected previously in bovine MFGM glycoproteins are included in Asn-215-linked oligosaccharides. The results show that the glycosylation of butyrophilin occurs in a site-specific manner.View full abstractDownload PDF (2453K) -
Takao Ojima, Hiroyuki Tanaka, Kiyoyoshi Nishita1995 Volume 117 Issue 1 Pages 158-162
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Mr 52, 000 subunit of Akazara scallop striated muscle troponin, which was tentatively identified as troponin I, was cleaved into two major fragments with CNBr: C-terminal 17 kDa fragment (CN17K) and N-terminal 35 kDa fragment (CN35K) [J. Biochem. 108, 519-521 (1990)]. CN17K inhibits rabbit reconstituted actomyosin Mg-ATPase activity, weakly in the absence of troponin T but strongly in its presence, together with Akazara tropomyosin. CN35K, however, hardly shows such inhibition. Thus, the amino acid sequence of the CN17K was determined by the Edman method. CN17K comprises 135 amino acid residues and its calculated molecular mass is 15, 732 Da. A computer search of the SWISS-PROT data base revealed the TnIs of crayfish tail muscle, rabbit skeletal muscle, and bovine cardiac muscle to be homologous proteins with total sequence homologies of 39, 30, and 30%, respectively, to CN17K. Significantly high homology was observed among these TnIs in the regions around residues 75-95, 99-114, and 135-151 of the rabbit TnI. From these facts, we conclude that the 52K subunit is a TnI.View full abstractDownload PDF (1842K) -
Tadayoshi Imai, Satoshi Okuda1995 Volume 117 Issue 1 Pages 163-168
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel type of pyridine nucleotide, containing two adenosine triphosphate ribose residues rather than one, was isolated from Azotobacter vinelandii strain O. The nucleotide was shown to be 2''- or 3''- (2'-phosphoadenosine-5'-diphosphoribosyl) nicotinamide adenine dinucleotide phosphate, in which 2'-phospho-5'-diphosphoadenosylribose was glycosidically linked to the NADP at position 2' or 3' of the nicotinamide mononucleotide moiety. The ATPribosylNADP did not show coenzyme activity for yeast glucose 6-phosphate dehydrogenase, nor was it cleaved by Neurospora crassa NAD (P) glycohydrolase, indicating that the biological properties conferred on the β-NADP molecule were largely modified by the attachment of the ATP-ribose group.View full abstractDownload PDF (1337K) -
Yasushi Nagata, Fumi Tashiro, Shigehiro Yi, Tatsuhumi Murakami, Shuich ...1995 Volume 117 Issue 1 Pages 169-175
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo ascertain whether a 6-kb upstream region of the human transthyretin (TTR) gene contains the cis-element (s) required for proper specificity and level of expression, transgenic mice carrying the human mutant TTR gene containing either 6-kb (6.0-hMet3O) or 0.6-kb (0.6-hMet30) of the upstream region were produced and studied. The 6.0-hMet30 gene was expressed in the yolk sac, liver, and choroid plexus, where the mouse endogenous TTR gene is also expressed. In contrast, expression of the 0.6-hMet30 gene was restricted to the yolk sac and liver. The expression levels of the 6.0-hMet30 gene in the liver and serum were similar to those of the mouse TTR gene, and about 10-fold those of the 0.6-hMet30 gene. Before birth, the developmental profiles of the expression of both transgenes in each tissue were similar to those of the mouse TTR gene. However, the expression levels of the 6.0-hMet30 gene in the liver and serum increased after birth to reach adult levels at an age of 4 weeks, while expression of the 0.6-hMet30 gene remained at a low level after birth. These results suggest that the 6-kb upstream sequence contains the cis-elements required for developmental, tissue-specific, and quantitatively normal expression.View full abstractDownload PDF (4275K) -
Binding Mode of Phospholipase A2 with a New Type of Phospholipid Analog Having an Oxazolidinone RingTakeshi Tani, Shinobu Fujii, Seiji Inoue, Kiyoshi Ikeda, Seiji Iwama, ...1995 Volume 117 Issue 1 Pages 176-182
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInhibition of phospholipases A2 (PLA2s) by a new type of monodispersed phospholipid analog, 3-dodecanoyl-4-phosphatidylcholinohydroxymethyl-2-oxazolidinone (oxazolidinone-PC), was investigated by the pH stat assay method using monodispersed 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC) as the substrate. The PLA2s used were those from bovine pancreas and cobra (Naja naja atra) venom (Group I) and from Japanese mamushi (Agkistrodon halys blomhoffii) venom (Group II). This new-type substrate analog was shown to inhibit competitively both types of venom and bovine pancreatic enzymes by binding to the active site in a similar manner to the carboxamide-type analog 2-dodecanoyl-amino-1-hexanol-phosphocholine (amide-PC). The binding of a stereoisomer, (R) -amide-PC, to N. naja atra (Group I) and A. halys blomhoffii (Group II) PLA2s was facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of (R) -oxazolidinone-PC to the N. naja atra (Group I) enzyme was found to be independent of Ca2+ binding, while its binding to the A. halys blomhoffii (Group II) enzyme was markedly facilitated by the binding of Ca2+ to the enzyme. The binding of (R)-amide-PC to N. naja atra PLA2 (Group I) was markedly influenced by the ionization state of the catalytic residue His 48, whereas the binding of (R)-oxazolidinone-PC was found to be practically independent of the ionization state of this residue. The Ca2+ dependency and participation of the catalytic group His 48 in the binding of genuine substrate to both types of PLA2s were found to be very similar to those for the oxazolidinone-PC, but differed greatly from those for the amide-PC, indicating that the binding mode of oxazolidinone-PC is very similar to that of the genuine substrate, but very different from that of the amide-PC.View full abstractDownload PDF (833K) -
Tomiko Asano, Haruo Shinohara, Rika Morishita, Ikuo Norota, Kanefusa K ...1995 Volume 117 Issue 1 Pages 183-189
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the functional relevance of the G-protein G0 to adenosine-induced effects in the heart, we studied the localization of G0 and its interaction with A1 adenosine receptors. Concentrations of G0 in various mammalian hearts differed markedly between the atrial and the ventricular muscle, as well as among species. In most species examined, the concentration of G0 was much higher in the atrium than in the ventricle. The highest levels of G0 in atria and ventricles were found in the ferret heart. An immunohistochemical study of the ferret heart with G0α-specific antibodies showed that G0 was localized throughout the membranes of cardiac myocytes, including the intercalated disks. In addition, G0 was densely distributed in the nerve fibers and Purkinje fibers. Analyses of G0α subtypes showed that bovine atrium mainly contained G0Aα, while bovine ventricle contained only G0Bα. By contrast, ferret ventricle contained both subtypes of G0α. To study the coupling of G0 to A1 adenosine receptors, receptors in ventricular membranes of ferrets, which had been pretreated with pertussis toxin, or purified receptors were reconstituted with purified G0A and G0B. The reconstitution experiments indicated that both subtypes of G0 coupled with A1 adenosine receptors. These results suggest that the effect of adenosine is mediated by both G0A and G0B in membranes of ferret cardiac myocytes.View full abstractDownload PDF (3209K) -
Yoshiharu Ohoka, Kenji Kontani, Taroh Iiri, Hiroshi Nishina, Toshiaki ...1995 Volume 117 Issue 1 Pages 190-196
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRetinoic acid-induced differentiation of human leukemic HL-60 cells is accompanied with the early induction of an ecto-enzyme of NAD+ glycohydrolase (NADase), which has recently been identified as human leukocyte cell surface antigen CD38 [Kontani, K. et al. (1993) J. Biol. Chem. 268, 16895-16898]. The terminal cell differentiation attendant upon the cell growth arrest was, but the early induction of CD38 NADase activity was not, inhibited by prior treatment of HL-60 cells with pertussis toxin, which catalyzed ADP-ribosylation of the membrane-bound αβγ-trimeric GTP-binding proteins. The prior treatment was, however, not essential for the toxin-induced inhibition of the cell differentiation; the inhibition by the addition of pertussis toxin was still observed even after retinoic acid-induced expression of CD38 antigen. This suggested that a pertussis toxin-sensitive mechanism was involved in a late process of cell differentiation. Indeed, HL-60 cells appeared to secrete a differentiation-supporting factor in response to retinoic acid, since the cell differentiation was accelerated and potentiated upon culture of the cells in a conditioned medium prepared from retinoic acid-treated cells. The action of the differentiation-supporting factor was destroyed by heating and markedly attenuated in pertussis toxin-pretreated HL-60 cells. Thus, the whole process of the retinoic acid-induced cell differentiation appeared to consist of two distinguishable periods in terms of sensitivity to pertussis toxin; the toxin-insensitive early period characterized by the induction of CD38 NADase activity and the toxin-sensitive late period in which the secretion of a differentiation-supporting factor might be involved.View full abstractDownload PDF (921K) -
Yo Kikuchi, Kyoko Suzuki-Fujita1995 Volume 117 Issue 1 Pages 197-200
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe synthesized a chimeric RNA between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a model substrate of the enzyme. The model substrate is the smallest substrate of RNase P, having a simple stem-loop structure. This model substrate was added to the 3'-end of M1 RNA. This chimeric molecule, which we call M1 RNA-MS, is a self-cleaving RNA and is cleaved much more efficiently than the M1 RNA-pre-tRNA, an artificial self-cleaving RNA previously synthesized [Kikuchi et al. (1993) Nucleic Acids Res. 21, 4685-4689], that consists of a full-size tRNA precursor and the M1 RNA. The self-cleavage of M1 RNA-MS at 10mM Mg2+ was an intramolecular reaction (cis-cleavage). Ca2+ supported the self-cleavage of M1 RNA-MS as effectively as Mg2+, although the self-cleavage of M1 RNA-pre-tRNA proceeded with low efficiency in the presence of Ca2+ as the only metal ion. Future application of the M1 RNA-MS molecule to the in vitro evolution of the M1 RNA and other experiments is proposed.View full abstractDownload PDF (2330K) -
Yuuki Kawamura, Hideaki Kume, Yoshiharu Itoh, Satoshi Ohtsuka, Sumiko ...1995 Volume 117 Issue 1 Pages 201-207
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConnectin (titin) links the Z line to the myosin filament in sarcomeres of vertebrate skeletal muscle. An 800 kDa fragment of α-connectin runs from the Z line up to the N2 line region in the I band, and the following β-connectin portion runs up to the edge of the M line on the myosin filament in chicken breast muscle sarcomeres. Immunoelectron microscopy showed that a 400 kDa fragment following the 800 kDa fragment reaches the edge of the myosin filament and, thereafter a 1, 700 kDa fragment runs to the M line on the myosin filament in chicken breast muscle sarcomeres. When stretched, the epitopes to anti-400 kDa fragment antibodies outside the myosin filament moved toward the inside of the I band.View full abstractDownload PDF (5366K) -
Ken Kainuma, Tstsuya Ookura, Yukio Kawamura1995 Volume 117 Issue 1 Pages 208-215
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein disulfide isomerase (PDI), which catalyses the folding of newly synthesized or denatured proteins through correct disulfide formation, was purified from soybean (Glycine max). The enzyme was purified 12, 000-fold over crude extracts to apparent homogeneity in six purification steps: 60-70% ammonium sulfate fractionation, and chromatography on DEAE Toyopearl 650M, Q-Sepharose Fast Flow, Hiload Superdex 200 pg, Phenyl Sepharose HP, and TSK G-3000 SW. The native enzyme had a molecular weight of 120 kDa on gel filtration. Subunit molecular weight was estimated as 63 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus indicating the enzyme to be comprised of two identical subunits. The enzyme pH optimum was 8.0 with reactivation of scrambled RNase, and the pI 7.65. The N-terminal amino acid sequence of soybean PDI was homologous to that of mature alfalfa as deduced from the cDNA sequence. Two identical active site sequences, APWCGHCK, were obtained from different proteolytic peptide fragments of soybean PDI. Soybean PDI facilitated reactivation not only of scrambled RNase, but denatured and reduced lysozyme and the Bowman Birk soybean trypsin inhibitor as well. This is the first report to appear on the the purification, characterization and amino acid sequence analysis of the active site of a plant PDI.View full abstractDownload PDF (1822K) -
Arco Y. Jeng, Mary Wong, Timothy Duelfer, Steven D. Shapiro, Richard A ...1995 Volume 117 Issue 1 Pages 216-221
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse macrophage metalloelastase was expressed in Escherichia coli. This recombinant enzyme (rMME) was present in the inclusion bodies that were solubilized in 7M guanidine HCl. After removal of guanidine HCl, rMME was purified with a Q-Sepharose column. Degradation of [3H]elastin by rMME absolutely required Ca2+; the optimal Ca2+ concentration was 5mM. NaCI stimulated the enzyme activity; maximal stimulation was obtained at 400mM. The rMME activity was inhibited by metalloprotease inhibitors, but not by serine, aspartyl, or thiol protease inhibitors. Among the divalent cations tested, only Ba2+ and Sr2+ exhibited marginal stimulation of rMME activity in the absenee of Ca2+. Cu2+, Zn2+, or Cd2+ strongly inhibited rMME activity with IC50 values between 68 and 180 μM, while Mg2+, Ba2+, Mn2+, Co2+, and Sr2+ had no effect. The requirement of Zn2+ for rMME activity was determined. Significant enzyme activity was present in rMME treated with EDTA followed by Q-Sepharose column chromatography. Only when the inclusion bodies were solubilized in the presence of 20mM EDTA, did an enzyme preparation which was absolutely dependent on exogenous Zn2+ for activity result. The optimal Zn2+ concentration for rMME activation was 100 μM. These results indicate that Zn2+ is tightly bound to rMME.View full abstractDownload PDF (1603K) -
Naoki Matsumoto, Somei Kojima, Toshiaki Osawa, Satoshi Toyoshima1995 Volume 117 Issue 1 Pages 222-229
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lymphocyte-specific protein, p50, is phosphorylated on Ser and Thr residues in mitogen-activated T cells, suggesting that this molecule plays some role in the T cell activation cascade. p50 was identified as lymphocyte specific protein 1 (LSP1), which is a putative calcium-binding protein. In the present study, to clarify the role of p50 protein in the cascade, in vivo and in vitro phosphorylation of this molecule, and the effect of the phosphorylation on its distribution in activated T cells were examined. First, to obtain a sufficient amount of p50 as a phosphorylation substrate, p50 cDNA, which encodes a protein of 330 amino acid residues with a molecular mass of 36, 728 Da, was cloned from an ICR mouse thymocyte cDNA library and expressed in Escherichia coli. When the putative coding region of p50 cDNA was expressed in E. coli, the product showed an apparent molecular mass of 50 kDa on SDS-PAGE. The recombinant p50 was phosphorylated in vitro by rabbit protein kinase C (PKC) and by murine cytosolic protein kinase, that was activated by a combination of phosphatidylserine and diacylglycerol. Furthermore, p50 was shown to be phosphorylated on the same sites in T cells upon stimulation with Con A as when phosphorylated in vitro by rabbit PKC, indicating that p50 is phosphorylated by PKC in Con A-stimulated T cells. On subcellular fractionation followed by immunoblotting analysis, membrane-bound p50 was shown to be released from the membrane following activation of PKC in T cells. These results and the recent finding that p50 binds to actin fibers raise the possibility that p50 controls the binding of actin fibers to the plasma membrane under regulation by PKC in T cells.View full abstractDownload PDF (4904K)
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