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Volume 117 (1995)
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Volume 117, Issue 6
Displaying 1-28 of 28 articles from this issue
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Exploring the Nucleotide-Binding Site in Proteins by Affinity Labeling and Site-Directed MutagenesisToshio Fukui1995 Volume 117 Issue 6 Pages 1139-1144
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAffinity labeling with nucleoside polyphosphopyridoxals, especially those with 3 or 4 phosphate groups, was effective for identifying the lysyl residue(s) located at or near the binding site for ATP, GTP, UDP-Glc, or ADP-Glc in various proteins. Furthermore, kinetic analysis of the mutant enzymes, in which the labeled lysyl residue was replaced with another amino acid by site-directed mutagenesis, provided evidence of its functional role. Affinity labeling of the mutant enzymes was useful for further identification of the hidden lysyl residue, which is unreactive in the wild-type enzyme but catalytically important. Comparison of the results of affinity labeling with different substrate analogues provided the information on the location of the labeled lysyl residue around the bound substrate. The affinity labeling reflected structural features of proteins, including their conformational flexibility.View full abstractDownload PDF (2049K) -
Kazunori Kataoka, Issei Hisamitsu, Naoko Sayama, Teruo Okano, Yasuhisa ...1995 Volume 117 Issue 6 Pages 1145-1147
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom the standpoints of long-term stability and simple handling, a glucose sensor composed of totally synthetic sensing moieties should be of great advantage. This study was performed to demonstrate the feasibility of a novel glucose sensing system in an aqueous milieu based on a change in the fluorescence on competitive binding between a fluorescent diol compound and glucose toward a phenylborate compound. 6, 7-Dihydroxy-4-methyl-coumarin (DHMC) was selected as a fluorophore, and 3-propionamidophenylboronic acid (PAPBA) as a phenylborate compound. The relationship between the glucose concentration and fluorescence intensity was linear in the range of 0 to 500mg/dl of glucose. The availability of various derivatives of DHMC and phenylborate compounds allows the incorporation of these functionalities on the tip of an optical fiber to construct an opto-sensing system for glucose.View full abstractDownload PDF (340K) -
Sakae Kitada, Kunitoshi Shimokata, Takuro Niidome, Tadashi Ogishima, A ...1995 Volume 117 Issue 6 Pages 1148-1150
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial processing peptidase (MPP) consists of α- and β-subunits (α-MPP and β-MPP). β-MPP has a putative metal-binding sequence (HXXEH). To determine whether the sequence of β-MPP is essential for the enzymatic activity, we individually mutated the histidines and glutamic acid to arginines and glutamine, respectively. The wild-type and mutated β-MPPs were co-expressed with α-MPP in Escherichia coli. All three mutants had completely lost the activity, whereas the lost activity was recovered on the addition of wild-type β-MPP. The activity of the wild-type enzyme was reduced by the mutant β-MPPs. We conclude from these observations that the HXXEH region is involved in the formation of the active site and that β-MPP is the catalytic subunit of MPP.View full abstractDownload PDF (1562K) -
Hideaki Tanaka, Ken-ichi Hayashi, Takahisa Akatsuka, Jiro Toyama, Kazu ...1995 Volume 117 Issue 6 Pages 1151-1155
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe morphologies of stearic acid and cytochrome c (cyt.c)-adsorbed stearic acid monolayers were investigated by Brewster angle microscopy (BAM) with various molecular areas of stearic acid. With an area of more than 0.38 nm2/molecule, many blight island domains and some bright circles were observed in the BAM image of the stearic acid monolayer. The blight site part became to occupy all the surface with compression, and then became more closely packed with an area of 0.22 nm2/molecule. On the other hand, a different BAM image was obtained for the cyt.c-adsorbed stearic acid monolayer, as follows: (i) a striped pattern was only observed in the presence of cyt.c; (ii) the number of bright circles in the presence of cyt.c was less than that in its absence. Furthermore, when a uniform BAM image was observed for the stearic acid monolayer with cyt.c, the intensity of the absorbance at 409 nm of cyt.c was the highest. By calculating the amount of cyt.c adsorbed on a stearic acid monolayer from the absorbance value, it was shown that cyt.c was most closely packed when an uniform SAM image was observed. These results suggest that the use of BAM and visible absorption spectroscopy together is useful for studying the morphology of a monolayer.View full abstractDownload PDF (2037K) -
Katsuya Kobayashi, Kazuhiko Takahashi, Shigeharu Nagasawa1995 Volume 117 Issue 6 Pages 1156-1161
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen stimulated with an insoluble immune complex consisting of tetanus toxoid and human anti-tetanus toxoid IgG antibody, human neutrophils recognized the immune complex via Fc receptors (FcγRII and FcγRIIIB) on their plasma membranes, resulting in Ca2+ accumulation, and finally phagocytosed the immune complex. Ca2+ depletion experiments suggest that Ca2+ accumulation is essential for the maximal phagocytic activity. A tyrosine kinase inhibitor, genistein, inhibited the phagocytosis of immune complex. Genistein also inhibited both the later prolonged Ca2+ accumulation below 100 μM, and the initial rapid Ca2+ accumulation above 100 μM. Stimulation of human neutrophils by immune complexes through FcγRs caused tyrosine phosphorylation of multiple neutrophil proteins within 15 s, proceeding to an increase in the intracellular Ca2+ concentration. A similar pattern of tyrosine phosphorylation was observed when FcγRII or FcγRIIIB was aggregated by an anti-FcγR monoclonal antibody and anti-mouse Ig. These results suggest that tyrosine phosphorylation mediates, at least in part, the later responses (Ca2+ accumulation and phagocytosis) of the cells and is an important early step in signaling via FcγRs.View full abstractDownload PDF (2907K) -
Shinpei Yamada, Kennichi Hojo, Hisashi Yoshimura, Katsutoshi Ishikawa1995 Volume 117 Issue 6 Pages 1162-1169
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe multicatalytic endopeptidase complex (20 S proteasome) is a latent high-molecular-mass multisubunit proteinase. In many investigations, SDS has been used as a proteasome activator at some fixed concentration that was apparently optimal. This study examined the effects of various divalent cations on the SDS-dependent peptidase and casein degradation activities of 20 S proteasome purified from Xenopus laevis oocytes at a series of SDS concentrations and the correlation between these effects and the critical micelle concentration (CMC) of SDS. Surprisingly, it was found that divalent cations such as Mg2+ markedly shifted the SDS-dependent activation profiles to a lower concentration range. Ca2+, Mn2+, Co2+, and Zn2+ also markedly reduced the optimum SDS concentration in the Suc-Leu-Leu-Val-Tyr-MCA hydrolysis reaction: for example, 5mM Co2+ reduced the optimum SDS concentration from 0.065 to 0.005%. However, in all cases examined the optimum concentrations were below the CMC. Cu2+, Hg2+, and Cd2+ strongly inhibited the SDS-dependent maximum activity without remarkably shifting the optimum SDS concentration. No correlation between the shift and the inhibition was recognized. Most interestingly, remarkable activation of casein degradation by SDS was observed only by addition of the divalent cations Mg2+, Ca2+, and Mn2+. These cations might be essential for casein degradation. The activation and inactivation ranges of SDS concentration varied with the species of substrate. These results suggested that cations have two independent effects on the reaction of 20 S proteasome: one is to modify the SDS-dependent activation profile, perhaps by inducing a conformational change of the enzyme that allows easier access of monomeric species of SDS to some site(s), resulting in activation or inactivation; and the other is an inhibitory effect on the reaction.View full abstractDownload PDF (2195K) -
Tetsumi Konishi, Yuji Karasaki, Minoru Nomoto, Hisamitsu Ohmori, Kiyot ...1995 Volume 117 Issue 6 Pages 1170-1177
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous work demonstrated a simultaneous induction of Hsp70 and nucleolin mRNA during the prereplicative stage of hepatocytes after partial hepatectomy [Ohmori, H. et al. (1990) Exp. Cell Res. 189, 227-232]. In the present study, changes of intracellular localization of these proteins were examined. Nucleolin, which mainly localized in the nucleus, increased with time and accumulated in the nucleolus around 12-18h after partial hepatectomy. Hsp70 protein also increased slightly around 6-12h after the operation. In accordance with this increase, immunohistochemical staining revealed that almost all nuclei of hepatocytes became Hsp70-positive, although Hsp70 was seen to be dispersed throughout the nucleoplasm and nucleolus at all times examined. Next, we isolated a cDNA clone of ribosomal protein (S-17) and examined its behavior. Induction of S-17 mRNA was observed to be essentially similar to that of nucleolin mRNA in regenerating rat liver, although inductions of histone H2A and H4 occurred at a later time, that is, in parallel with DNA synthesis. Furthermore, we observed a simultaneous induction of Hsp70 and nucleolin mRNA by serum-stimulation after serum-depletion in HeLa and IAR-20 (rat) culture cells. These results suggest that the induction of Hsp70, in addition to nucleolin, was not fortuitous but may be involved in the early events of liver regeneration.View full abstractDownload PDF (3095K) -
Kouki Ono, Yukari Kawanaka, Yuri Izumi, Hiroshi Inui, Kazutaka Miyatak ...1995 Volume 117 Issue 6 Pages 1178-1182
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inducing effects of ethanol on alcohol dehydrogenase and the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, in Euglena cells were investigated. Ethanol as the sole carbon source resulted in increases in alcohol dehydrogenase and the two glyoxylate cycle enzymes. The experimental results indicated that ethanol is assimilated by alcohol dehydrogenase and the glyoxylate cycle in Euglena. Mitochondria from aerobically grown Euglena contain a unique type of alcohol dehydrogenase that accounts for their ability to respire with ethanol as a substrate. This alcohol dehydrogenase was purified to homogeneity from ethanol-grown Euglena gracilis. The mitochondrial alcohol dehydrogenase was NAD+-specific but not NADP+-specific. Ethanol was the most active substrate, but the enzyme was also active towards 1-butanol, 1-heptanol, cinnamyl alcohol, and myristyl alcohol. These results indicated that mitochondrial alcohol dehydrogenase participated in alcohol metabolism in Euglena gracilis.View full abstractDownload PDF (1558K) -
Shoko Kido, Yukio Doi, Fimi Kim, Emi Morishita, Hiroshi Narita, Shigen ...1995 Volume 117 Issue 6 Pages 1183-1191
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVitelline membrane outer layer protein I (VMO-I) tightly bound to ovomucin fibrils of hen's egg yolk membrane was characterized in terms of its amino acid sequence and structural stability. The deduced sequence of VMO-I using the conventional sequencing method is: RTREYTSVITVPNGGHWGKWGIRQFCHSGYANGFALKVEPSQFGRDDTALNGIRLRCLD-GSVIESLVGKWGTWTSFLVCPTGYLVSFSLRSEKSQGGGDDTAANNIQFRCSDEAVLVGD-DLSWGRFGPWSKRCKICGLQTKVESPQGLRDDTALNNVRFFCCK. Thus, VMO-I is composed of 163 amino acid residues with a calculated molecular weight of 17, 979. The sequence confirms the cDNA sequence of VMO-I we recently determined and does not show any significant similarity to proteins compiled in the NBRF database. Two of the four disulfide bonds found in VMO-I were estimated to lie between Cys26 and Cys57 and between Cys79 and Cys110. The sequence analyses show that VMO-I contains three 53-residue internal repeats that contain distinctive regions of turns flanked by β-sheets consistent with the recent finding that the molecule contains a new β-fold motif, the β-prism. The molecular characteristics of VMO-I in solution were examined by CD spectroscopy in the far and near ultraviolet regions, NMR spectroscopy, and high sensitive differential scanning calorimetry (DSC). CD spectra in the far UV region at room temperature were similar to that assigned to a random coil, while in the near UV region, small positive peaks were observed. The ellipticity in both regions decreased on raising the temperature. Proton NMR experiments showed the native structure unfolds to unordered conformations at 70°C. A single positive peak appeared in the DSC curve with the transition temperature of 67.5°C, from which an unfolding enthalpy of 107 kcal/mol and a heat capacity change of 2.6 kcal/mol/K were obtained. These results showed a normal degree of stability for the β-prism structure found in VMO-I.View full abstractDownload PDF (1088K) -
Kou-Wha Kuo, Long-Sen Chang, Chun-Chang Chang1995 Volume 117 Issue 6 Pages 1192-1195
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and convenient method for determining the binding activity of α-neurotoxins toward soluble nicotinic acetylcholine receptor (snAChR) by precipitation with DEAE-Sephacel was established. The determination was carried out by incubation of 125I-neurotoxin with snAChR, followed by precipitation with DEAE-Sephacel. The DEAE-Sephacel particles bind negatively charged snAChR with high affinity and simultaneously precipitate the 125I-neurotoxin bound to the receptors. After centrifugation, the free 125I-neurotoxin in the supernatant was counted, and the amounts of neurotoxins bound to snAChR could be determined. Two α-neurotoxins, cobrotoxin and α-bungarotoxin, were employed to verify the feasibility of this determination. The different binding characteristics of cobrotoxin and α-bungarotoxin to snAChR could be distinguished. This method required only small quantities of DEAE-Sephacel (7mg), snAChR (0.54 μg), and 125I-neurotoxin (90 fmol) for each reaction, and minimized the handling of isotopic materials as compared with the conventional methods. This method is reliable, reproducible, and superior to current methods for the determination of the snAChR-binding activity for α-neurotoxins.View full abstractDownload PDF (470K) -
Masato Yano, Kazutoyo Terada, Kenji Umiji, Katsura Izui1995 Volume 117 Issue 6 Pages 1196-1200
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphoenolpyruvate carboxylase (PEPC) [EC 4. 1. 1. 31] has a highly conserved and unique sequence, 578-FHGRGGSIGRGGAP-591 (on Escherichia coli, PEPC), in which a GRGG motif is repeated twice with two intervening residues. Since previous chemical modification studies suggested the functional importance of arginine residues, the invariant Arg587 in this region was replaced with Ser, and the enzymatic properties of the resulting mutant enzyme (R587S) were investigated. Replacement led to virtual loss of the catalytic activity to form oxaloacetate. The specific activity was 37 nmol•min-1•mg-1, which corresponds to 2×10-4-fold the activity of the wild-type enzyme. However, the activity of bicarbonate- and Mg2+-dependent hydrolysis of phosphoenolpyruvate (PEP) to pyruvate appeared for the mutant enzyme with a specific activity of 2.1 μmol•min-1•mg-1. In view of the stepwise reaction mechanism proposed for PEPC, this activity can be attributed to impairment of the subsequent partial reaction(s) following the formation of the intermediate carboxyphosphate. The half-saturation concentration (S0.5) of HCO3- in R587S was about 100-fold that in the wild-type enzyme, whereas the respective values for PEP and Mg2+ were 20- and 15-fold, indicative of this residue participating in the binding of HCO3-.View full abstractDownload PDF (717K) -
Hiroyuki Maeda, Tetsuya Inazu, Katsuya Nagai, Shingo Maruyama, Gizou N ...1995 Volume 117 Issue 6 Pages 1201-1208
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that p72syk, a non-receptor tyrosine kinase, was activated maximally at 10 s after thrombin or thromboxane A2 stimulation, even in platelets that were not allowed to aggregate [Taniguchi et al. (1993) J. Biol. Chem. 268, 2277-2279; Maeda et al. (1993) Biochem. Biophys. Res. Commun. 197, 62-67]. Then, the change in the shape of porcine platelets induced by the thromboxane A2 analogue, STA2, and the role of protein-tyrosine kinases including p72syk in this response were evaluated, using the shape-change parameter. We show that p72syk activation is correlated with the disc-sphere change in a time- and dose-dependent manner following stimulation by STA2. Tyrphostin B44, a potent protein-tyrosine kinase inhibitor, reduced the thromboxane A2-evoked p72syk activation and the disc-sphere change in a dose-dependent manner. Furthermore, the translocation of p72syk to the cytoskeleton-rich fraction and an increase in the tyrosine phosphorylation of an about 120 kDa protein were observed during the disc-sphere change induced by STA2. These lines of evidence suggest that the activation of protein-tyrosine kinases such as p72syk may be involved in the disc-sphere change response in thromboxane A2-stimulated porcine platelets.View full abstractDownload PDF (3948K) -
Byung Hyun Lee, Takashi Hibino, Tetsuko Takabe, Peter J. Weisbeek, Ter ...1995 Volume 117 Issue 6 Pages 1209-1217
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis. The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from -4 to +1. Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins. Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type. It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect. Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect. These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f. Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested. Based on these results, the electron transfer pathway from the heme to P700 via plastocyanin is discussed in relation to the role of charged amino acid residues.View full abstractDownload PDF (2034K) -
Hachiro I. Yamanaka, Yasuyuki Kirii, Hiroshi Ohmoto1995 Volume 117 Issue 6 Pages 1218-1227
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn improved combinatorial library system to raise mouse monoclonal antibodies was constructed. PCR primers have been newly designed to optimize the reaction for Pfu DNA polymerase, which has proofreading activity. The phagemid vector (pPDS) is designed to accommodate VH and VK cDNAs, which had previously been assembled by PCR either in single chain fragment of variable regions (scFv) or Fab form. Antibody cloned in scFv form can be converted to Fab form by substituting the scFv linker of (Gly4Ser)3 with a fragment containing murine CH1 cDNA. This vector will produce soluble Fab in non-amber suppressor cells and allow the shuffling of light chains against a heavy chain. Hybridoma cell lines producing anti-human procollagenase monoclonal antibodies were used as the source of antibody mRNA. Antigen-binding ability of both scFv- and Fab-displaying phage was confirmed by ELISA against human procollagenase. They were also analyzed by DNA sequencing to verify the fidelity of Pfu DNA polymerase and to identify the primer incorporated. The mutation rate was considerably reduced compared to the mutation rate achieved by Taq DNA polymerase. Primers are incorporated into target sequences in most cases.View full abstractDownload PDF (1746K) -
Mikio Tomida1995 Volume 117 Issue 6 Pages 1228-1231
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe recombinant soluble mouse D-factor/LIF receptor (sD-factor-R) was expressed in COS-7 cells. Scatchard analysis of the bindings of mouse 125I-D-factor and human 125I-D-factor to the sD-factor-R indicated dissociation constants (Kd) of 12 and 0.267 nM, respectively, which were comparable to those of the binding protein in mouse serum. The apparent molecular masses of the sD-factor-R and human D-factor observed by gel filtration chromatography were 150 and 50 kDa, respectively. The size of the sD-factor-R•human D-factor complex was approximately 200 kDa, indicating that D-factor forms a 1:1 complex with the sD-factor-R. The sD-factor-R inhibited the induction of differentiation of mouse myeloid leukemic M1 cells by mouse D-factor by blocking the binding of this factor to the cells.View full abstractDownload PDF (526K) -
Kazunori Anzai, Yasuyuki Yoshioka, Ken'ichi Hatanaka, Yasuhito Nakagaw ...1995 Volume 117 Issue 6 Pages 1232-1237
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe measured the translocation of exogenous radiolabeled phospholipid probes from the outer to the inner leaflet in the synaptosomal plasma membranes from the electric organs of Narke japonica, a Japanese marine ray. These radioactive probes can provide a convenient and highly sensitive means of measuring the translocation kinetics of phospholipids between outer and inner leaflets of biomembranes that are available only with low yield [Anzai et al. (1993) Biochim. Biophys. Acta 1151, 69-75]. Translocation kinetics revealed that the behavior of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidyleholine (PC) differed. PS quickly disappeared from the outer leaflet of the bilayer. The translocation of PE and PC was slow and about 80% of the PE- and PC-probes remained in the outer leaflet at 3h. These results differ from those of translocation in erythrocyte membranes measured using the same probes, where aminophospholipid translocase translocated both PS and PE from the outer to the inner leaflet of the bilayer, although the rate was faster for PS than for PE. In this study, the translocation was apparently PS-specific, and it was inhibited by SH reagent or intrasynaptosomal ATP-depletion. These findings clearly demonstrate that the translocation is driven by an ATP-dependent protein, which has apparent PS-specificity. We suggest that there is a translocase other than erythrocyte-type aminophospholipid translocase in synaptosomal plasma membranes from N. japonica.View full abstractDownload PDF (1963K) -
Yutaka Inaguma, Kaori Hasegawa, Sachiyo Goto, Hidenori Ito, Kanefusa K ...1995 Volume 117 Issue 6 Pages 1238-1243
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe levels of hsp27 and αB crystallin in various tissues of rats that had been subjected to heat stress were determined by specific immunoassays. When rats were immersed in a water bath at 42°C for 20min, the levels of hsp27 in most tissues, including central nervous tissue, liver, lung, spleen, adrenal glands, and hypophysis, had increased dramatically 8 to 16h after the treatment. αB crystallin was also induced at high levels in the liver and adrenals, but not much was induced in the central nervous tissue. The increases in the levels of both hsp27 and αB crystallin in response to heat stress were markedly inhibited when ethanol or an α1-adrenergic antagonist, prazosin, was administered before, but not after, the stress period. The expression of mRNA for hsp27 was suppressed in the livers of rats that had received ethanol or prazosin. A β-adrenergic antagonist, propranolol, and an α2-adrenergic antagonist, yohimbine, did not inhibit induction of the synthesis of the two proteins. Accumulation of hsp70 after heat stress, at least in the liver, was inhibited by ethanol or prazosin in a similar manner to that of hsp27, as detected on Western blot analysis. These results suggest that the induction of hsp27 and αB crystallin, as well as of hsp70, in tissues of heat-stressed rats is controlled by a physiological process(es) that is sensitive to ethanol and prazosin, and is operative for a short time during the application of heat stress.View full abstractDownload PDF (4207K) -
Keiichi Kamoshita, Mayumi Shiota, Masafumi Sasaki, Yasuhiro Koga, Yuus ...1995 Volume 117 Issue 6 Pages 1244-1253
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe recently purified the calcium-independent processing protease named viral envelope glycoprotein maturase (VEM), that converts human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor gp160 to gp120 and gp41, from the human CD4+ T cell line, Molt-4 clone 8 [Kido, H., Kamoshita, K., Fukutomi, A., and Katunuma, N. (1993) J. Biol. Chem. 268, 13406-13413]. In this report, we deal with the inhibitor specificity and calcium requirement for intracellular gp160 processing in cultured HeLa cells and human CD4+ lymphocytes. Processing of gp160 in these cells infected with recombinant vaccinia virus encoding the gp160 gene was not affected by intracellular calcium depletion induced by the calcium ionophore A23187 and EGTA or by intracellular calcium administration. Processing of gp160 by the purified VEM in vitro was not inhibited by EDTA, EGTA, or the metallo-protease inhibitor phosphoramidon, but was specifically inhibited by a substrate analog, decanoyl-RVKR-chloromethylketone, and the trypsin-type protease inhibitors aprotinin, HI-30, and diisopropyl fluorophosphate (DFP). It was also inhibited by E-64 and thiol reagents. But intracellular gpl60 processing was inhibited only by permeable, low molecular mass inhibitors of VEM, such as DFP, E-64, and thiol reagents. Syncytium formation induced by cell surface gp120 was also inhibited by permeable inhibitors of VEM. Taken together, our results indicate that calcium ions may not be essential for intracellular gp160 processing and so HIV-1 gp160 induced by recombinant vaccinia virus may be processed mainly by a protease(s) that does not require calcium ions, such as VEM in these cells.View full abstractDownload PDF (7317K) -
Seiji Kawazoye, Sheng-Fang Tian, Shuji Toda, Toshinobu Takashima, Tosh ...1995 Volume 117 Issue 6 Pages 1254-1260
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSodium dodecyl sulfate (SDS), an anionic hydrophobic ligand, is known to alter the mechanical properties of elastic fibers. In order to analyze the mechanism of the alteration, two forms of fibrous elastins, “solid” and “powder” elastins, which consisted of fascicular elastic fibers and single or oligomeric elastic fibers, respectively, were prepared from bovine aorta, and the interactions of SDS with these elastins in the presence and absence of 0.15M NaCl were studied. The solid elastin was able to retain 1.2- to 1.4-fold larger amounts of SDS than the powder elastin under both conditions, and both elastins retained 1.2-fold or larger amounts of SDS in the presence of NaCl than in its absence. Whereas both elastins released the retained SDS gradually on repeated washing with an SDS-free buffer, the release rates from the solid elastin, especially the rate in the presence of NaCl, were much smaller than those from the powder elastin, and the solid elastin retained approximately 40% of the bound SDS under conditions where the powder elastin lost most of its SDS. The SDS-binding capacities of both elastins were significantly lower than those of soluble κ-elastin and serum albumin, which bound SDS homogeneously on the polypeptide chains. When the washed SDS-bound solid elastin was incubated with methylene blue and examined under a microscope, most of the methylene blue-SDS complex was located at the interfiber spaces of the elastic fibers. These results suggest that SDS alters the mechanical properties of elastic fibers by binding to the interfiber spaces and surfaces of the fibers rather than by binding to the internal polypeptide chains.View full abstractDownload PDF (1945K) -
Javier Fernandez, Anthony A. Dimarco, L. Nicholas Ornston, Shigeaki Ha ...1995 Volume 117 Issue 6 Pages 1261-1266
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS4-Hydroxybenzoate 3-hydroxylase [EC 1. 14. 13. 2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their reponse to C1- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined.View full abstractDownload PDF (787K) -
Kazumasa Ohashi, Shigeyuki Honda, Noriko Ichinomiya, Toshikazu Nakamur ...1995 Volume 117 Issue 6 Pages 1267-1275
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously identified a human cDNA encoding a novel receptor tyrosine kinase, termed Sky, which is predominantly expressed in the brain and has a unique extracellular domain consisting of two immunoglobulin (1g)-like and two fibronectin type III (FN III) motifs. In attempts to define the functional role of the Sky receptor, we cloned a rat sky cDNA, and localized the sites of expression of sky transcript in the adult rat brain by Northern blot and in situ hybridization analyses using the cloned rat cDNA as a probe. The deduced amino acid sequence of rat Sky has an overall sequence and a domain topology highly conserved with human Sky (90% overall identity and 98% identity within the tyrosine kinase domain). Northern blot analysis revealed that a single 3.8-kb sky mRNA is expressed in PC12 pheochromocytoma and Neuro-2a neuroblastoma cell lines and in various regions of the adult rat brain. In situ hybridization analysis revealed widespread but confined neuronal populations in adult rat brain that express sky transcript; prominent hybridization signals were detected in the inner granular layer of the olfactory bulb, CA-1 area of the hippocampus, granule cell layer of the cerebellum, tenia tectum and cingulate gyrus neurons, and wide regions of cortex layers II-VI. The high level of expression of sky mRNA in neurons in restricted brain regions suggests that the Sky receptor may play an important role in development, function, and maintenance of specific neuronal populations in the central nervous system.View full abstractDownload PDF (4522K) -
Jianguo Gu, Yoshinao Wada1995 Volume 117 Issue 6 Pages 1276-1279
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCarbohydrate-deficient glycoprotein syndrome (CDGS) is a congenital disorder characterized by neurological and developmental defects. We have examined the expressions of the small proteoglycans decorin and biglycan in cultured skin fibroblasts from a patient with CDGS Type-I. Northern blotting analysis identified a marked reduction in decorin mRNA and an increase in biglycan mRNA levels. The decorin protein in the culture medium was decreased. Responses to interleukin-1 β (IL-1 β) and transforming growth factor-β 1 (TGF-β 1) were apparently abnormal; decorin was only slightly up-regulated by IL-1 β, while biglycan was markedly down-regulated by IL-1 β and significantly up-regulated by TGF-β 1. The constitutional and developmental abnormalities characteristic of CDGS may be associated with such derangements in the expression of proteoglycan genes.View full abstractDownload PDF (1475K) -
Kazuaki Yokoyama, Ichiro Kudo, Keizo Inoue1995 Volume 117 Issue 6 Pages 1280-1287
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn “A1 type” phospholipase activity with serine-phospholipid preference was released by rat activated platelets. It was distinct from the secretory type II phospholipase A2 [Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101, 625-631] and co-purified with the secretory lysophosphatidylserine-selective lysophospholipase activity [Higashi, S., Kobayashi, T., Kudo, I., and Inoue, K. (1988) J. Biochem. 103, 442-447]. Several lines of evidence indicated that a single protein was responsible for the phospholipase A1 and lysophospholipase activities. Marked accumulation of lysophospholipids was observed in rat calcium ionophore-activated washed platelets and both phospholipase A1/lysophospholipase and type II phospholipase A2 were shown to contribute to this phospholipid degradation. A selective inhibitor of type II phospholipase A2 reduced the phospholipid degradation and enhanced the clotting time and prothrombinase activity. These results indicate that secretory platelet phospholipases may play a role in regulation of blood clotting.View full abstractDownload PDF (965K) -
Nurith Mador, Amos Panet, David Latchman, Israel Steiner1995 Volume 117 Issue 6 Pages 1288-1297
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHerpes simplex virus type 1 (HSV-1) is transcriptionally active during latent infection in human peripheral sensory ganglia. This transcription has been linked to the ability of the virus to reactivate, but its potential gene products and mechanisms of action are unknown. To analyze the viral latency-related transcripts in neuronal and non-neuronal cell lines in an isolated cellular system, a 10.4 kb DNA fragment, which covers the entire viral transcriptionally active latency-associated region, was cloned under control of the constitutive cytomegalovirus promoter (pNM3). During transient transfection of a human embryonic kidney 293 cell line, pNM3 expressed high levels of the 2.0 kb latency-associated transcript (LAT) that was not polyadenylated. The 1.5 kb LAT as well as the minor hybridizing RNAs could not be identified by Northern blotting analysis. pNM3 expression was further analyzed following transfection of two neuronal, C 1300 and ND7 cell lines. The 2.0 kb LAT was synthesized at high levels in these cell lines. The 1.5 kb LAT, which in vivo can be identified only during HSV-1 latent infection in tissues which facilitate reactivation, was present at very low amounts in 293 and C 1300 cells using reverse transcription PCR analysis. Higher amounts of the 1.5 kb LAT were produced in ND7 cells, a neuronal cell line shown to possess neuronal-specific splicing proteins. However, the 1.5 kb LAT was present in ND7 cells in lesser amounts than produced during latent infection in peripheral sensory ganglia. This novel cellular system provides now a tool for future studies of the role of the 1.5 kb and the 2.0 kb LATs in HSV-1 latency.View full abstractDownload PDF (4978K) -
Masao Iwata, Yasutada Imamura, Tasuku Sasaki, Toshihiko Hayashi1995 Volume 117 Issue 6 Pages 1298-1304
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe extracts from bovine lens capsule with acetic acid contained, after reduction, three major collagenous polypeptides with Mr=180k, 175k, and 160k, which were specifically immuno-stained with anti-type IV collagen polyclonal antibody. The biochemical properties of 180k and 160k polypeptides were akin and were distinct from that of 175k polypeptide [J. Biochem. (1993) 114, 358-362]. In the present study, evidence that the 160k and 180k polypeptides from bovine lens capsule both originated from α1(IV) was obtained on the basis of reactivity with a monoclonal antibody that recognizes α1(IV) chain at the collagenous sequence contained in [KGEPGLPGRGFPGFP]. The epitope-bearing sequence was identified from the following three experiments. Pepsin-solubilized polypeptides from human placenta were purified by affinity chromatography on the antibody-coupled column and sequenced. The restriction map of the clones positively reactive with the monoclonal antibody from human placenta cDNA library was superimposed on that of human α1(IV) cDNA at a specific region. Synthetic peptides corresponding to the sequence were assayed for inhibitory activity against the reaction between epitope-bearing pepsin fragments and the antibody. The 180k and 160k polypeptides showed similar intensities in protein staining as well as in immuno-staining with the monoclonal antibody. In contrast, the 175k polypeptide did not react with the monoclonal antibody, indicating that it is a genetically distinct type IV collagen chain, presumably α2(IV) from its abundance. The 160k, a major type IV collagen polypeptide, is a short form of α1(IV) present as a tissue form in bovine lens capsule.View full abstractDownload PDF (2953K) -
Masatomo Maeda, Seikyo Asahara, Tsuyoshi Nishi, Sotaro Mushiake, Toshi ...1995 Volume 117 Issue 6 Pages 1305-1311
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA DNA segment containing the 5'-upstream region and amino terminal reading frame of the gastric intrinsic factor gene was cloned from rat and its nucleotide sequence was determined. S1 mapping demonstrated that the transcription initiation site is located downstream of the second TATA-box sequence. Similar sequence motifs to those in the pepsinogen genes transcribed in gastric chief cells were found in the deduced sequence, suggesting that the rat intrinsic factor gene is transcribed in these cells. The genes for the intrinsic factor and its homologous protein transcobalamin I were apparently derived from a common ancestoral gene, since the positions of their intron insertions as well as the amino acid residues are conserved. Northern blot hybridization showed that the gene for the intrinsic factor is transcribed in the stomach but not detectably in the intestine, kidney, testis, brain, heart, liver, lung, or spleen. In situ hybridization using radioactive comple-mentary RNA clearly indicated that the major transcription site in gastric glands is chief cells. Different locations of expression of intrinsic factor proteins in various mammals were observed previously using antibodies: in rat parietal cells and chief cells, in mouse chief cells, and in human parietal cells. The present results clearly demonstrated the intrinsic factor mRNA mainly in chief cells of adult rats, as in mice, suggesting that transcriptional regulation of the intrinsic factor gene is essentially the same in rodents.View full abstractDownload PDF (3211K) -
Juan Alvarez-Bravo, Shoichiro Kurata, Shunji Natori1995 Volume 117 Issue 6 Pages 1312-1316
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we reported that a synthetic undecapeptide, KLKLLLLLKLK-NH2, and its D-enantiomer have potent bactericidal activities against both Gram-positive and Gramnegative bacteria. Here we examined the mode of action of KLKLLLLLKLK-NH2 with special reference to its effect on bacterial membranes. We found that both the outer and inner membrane of Escherichia coli become permeable to low molecular mass substances when treated with this peptide. Under these conditions, the bacteria lost the ability to synthesize ATP and to transport praline, suggesting that their electrochemical membrane potential was disrupted. This peptide appears to form numerous channels in bacterial membranes that interfere with membrane functions, resulting in cell death.View full abstractDownload PDF (2103K) -
Tatsuo Kurihara, Ji-Quan Liu, Vincenzo Nardi-Dei, Hiromoto Koshikawa, ...1995 Volume 117 Issue 6 Pages 1317-1322
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic dehalogenation of L-2-halo acids, with inversion of the C2-configuration. Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (36-70% identity), and they are supposed to catalyze the reaction through the same mechanism. To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid residues of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which consists of 232 amino acid residues, by replacement of D by N, E by Q, R by K, and vice versa, S and T by A, Y and W by F, M by L, and H by N. We found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the Km value for the substrate, showing their involvement in the catalysis. The roles of these residues are discussed.View full abstractDownload PDF (711K)
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