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Volume 94 (1983)
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- Issue 5 Pages 1353-
- Issue 4 Pages 1029-
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Volume 94, Issue 5
Displaying 1-42 of 42 articles from this issue
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Masahiko HIRAMATSU, Eiko IKEDA, Masanori KASHIMATA, Naomi MINAMI, Hiro ...1983 Volume 94 Issue 5 Pages 1353-1358
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of dibutyryl cyclic AMP (DBcAMP) and related compounds on collagen synthesis in a clonal osteoblast-like cell line, MC3T3-El, were investigated. The addition of DBcAMP to cultures increased the hydroxyproline content of the cells. It also enhanced the incorporation of labeled proline into collagen and elevated the activity of prolyl hydroxylase, an enzyme involved in collagen synthesis. These effects were observed at concentrations of 0.1 to 2mM DBcAMP. 8-Bromo cyclic AMP also increased the hydroxyproline content of the cells, while sodium butyrate and dibutyryl cyclic GMP had no such effect. These results suggest that the intracellular accumulation of cyclic AMP in osteoblasts leads to their active production of collagen, a major component of the organic matrix of bone.View full abstractDownload PDF (423K) -
Kyoko NAKAMURA, Toshio ARIGA, Tomoyasu YAHAGI, Tadashi MIYATAKE, Akemi ...1983 Volume 94 Issue 5 Pages 1359-1365
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSkeletal muscle gangliosides prepared from ten species of animals (human, monkey, bovine, hog, dog, cat, rabbit, guinea pig, mouse, and chicken) were studied by two-dimensional thin-layer chromatography. Densitometric quantification of spots on the chromatograms was carried out with an image analysis system equipped with a computer system. In about thirty-five resorcinol-positive spots, sixteen components could be identified by comparison with authentic standards we had previously obtained. GM3 ganglioside was found as a major component in all examined animals, but there were remarkable species variations in the minor gangliosides. In bovine and hog muscle gangliosides, more than fifteen minor components were detected owing to the presence of N-glycolyl neuraminic acid species. In contrast to this, muscle gangliosides of human, dog, cat, rabbit, and chicken showed relatively simple patterns because of the absence or the small amounts of N-glycolyl type gangliosides. The presence of lactosamine containing gangliosides was characteristic of muscle gangliosides of the examined species.View full abstractDownload PDF (1604K) -
Masafumi TSUJIMOTO, Keizo INOUE, Shoshichi NOJIMA1983 Volume 94 Issue 5 Pages 1367-1373
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and rapid purification method for human serum C-reactive protein (CRP) was developed. CRP was strongly adsorbed on a DEAE-cellulose column and was easily separated from other serum proteins. CRP was purified approximately 1, 000-fold with a high yield (50%). The final preparation showed a single band as judged by SDS-polyacrylamide gel electrophoresis, polyacrylamide gel disc electrophoresis, and polyacrylamide gel isoelectric focusing.
The fluorescence of the complex of CRP and 8-anilino-1-naphthalene sulfonate (ANS) changed with change of the pH, suggesting that CRP may show pH-dependent conformational change. This finding could account for the peculiar behavior of the protein in isoelectric focusing; it shows an isoelectric point of 7.4 when the starting pH is 7.0, whereas it shows two isoelectric points, 5.3 and 7.4, when the starting pH is 5.5. Ca2+-dependent change of the fluorescence of the complex of CRP and ANS was also detected. These results suggest a pH- and Ca2+-dependent conformational change of CRP.View full abstractDownload PDF (1179K) -
Iwao TABUSHI, Takako NISHIYA, Tatsuhiko YAGI, Hiroo INOKUCHI1983 Volume 94 Issue 5 Pages 1375-1385
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA detailed kinetic study on the successive four-step reduction of cyt c3, which has four heme units in a single protein, III4→III3II→III2II2→III II3→II4, was carried out by stopped-flow electronic spectroscopy (SF-UV) and stopped-flow circular dichroism spectroscopy (SF-CD). Based on the absorbance change vs. time and the ellipticity change vs. time at the characteristic CD, together with the electronic absorption of the enzyme, rate constants for the successive four electron transfer steps, k1-k4, were successfully estimated by computer simulation. The rate constants of the four steps (k1=19.8 s-1, k2=11.9 s-1, k3=8.9 s-1, and k4=1.6 s-1; 8.0 10-4M Na2S2O4) are quite different from the statistical values (4:3:2:1), thus excluding the possibility of random reduction of hemes of equal reactivities. Instead, each heme has its own reactivity, probably dependent on its local environment. The value of k3 is somewhat higher than the statistical value, indicating the existence of an autoacceleration effect, although small. This autoacceleration is most probably due to a unique heme-heme and/or heme-environment interaction since unusual CD and electronic absorptions were observed at 350-400 nm at about the time corresponding.View full abstractDownload PDF (696K) -
Akihiko MORIYAMA, Makoto SASAKI1983 Volume 94 Issue 5 Pages 1387-1397
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSuccinyltrialanine p-nitroanilide (STANA)-hydrolytic enzyme was purified 5, 200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS).
The pl of the enzyme was 4.9 by disc gel electrofocusing and the molecular weight was calculated to be 72, 000 by gel filtration on a Sephadex G-150 column and 74, 000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinylGly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succiny-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 μg/ml and 18.2 μg/ml, respectively. The enzyme was completely inhibited by 10-7 M p-chloromercuribenzoic acid (pCMB), 10-7 M p-chloromercuriphenylsulfonic acid (pCMPS), and 10-4 M diisopropyl phosphofluoridate (DFP), but not by 1mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3. 4. 21. 26].View full abstractDownload PDF (1396K) -
Kunihiko KONNO, Shuji KODAMA, Ken-ichi ARAI, Shizuo WATANABE1983 Volume 94 Issue 5 Pages 1399-1407
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The reactivities of scallop myosin with 5, 5'-dithiobis-(2-nitrobenzoate) (DTNB) and with 2, 4, 6-trinitrobenzene sulfonate (TNBS) were found to be affected by dissociation and association of regulatory light chains (RLC) of myosin. 2. Approximately 4 mol of sulfhydryl groups of “desensitized” myosin (DM) were masked on association of DM with RLC. When these sulfhydryl groups were reacted with DTNB, the modified DM became incapable of associating with RLC, but when the modified DM was treated with 2-mercaptoethanol, the ability to associate with RLC was fully recovered. 3. The DTNB-reactivity of scallop myosin and its RLC content were measured as a function of calcium and magnesium concentrations. The results thus obtained could be explained in terms of our previous suggestion (J. Biochem. 94, 1061 (1983)) that there are two different attachments between DM and RLC. 4. The relation between the TNBS-reactivity and the RLC content was not simple but complex. Not the extent, but the rate of trinitrophenylation of scallop myosin was affected by dissociation and association of DM with RLC; thus, the involved TNBS-reactive lysine residues did not seem to be in the regions on DM and RLC that would be physically covered upon DM-RLC association. 5. The amount of the involved lysine residues was estimated to be only 1 mol per mol of myosin. Modification of the specific lysine residues resulted in a partial decrease in the DM-RLC association.View full abstractDownload PDF (2669K) -
Shugo WATABE, Thi Ngoc-Loan DINH, Yoshihiro OCHIAI, Kanehisa HASHIMOTO1983 Volume 94 Issue 5 Pages 1409-1419
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive light chains were isolated from the ordinary and dark muscle myosins of mackerel Pneumatophorus japonicus japonicus, by a method consisting of DTNB and urea treatments, followed by DEAE-cellulose chromatography. Some physicochemical and immunochemical properties of the light chains thus obtained were analyzed.
1. A1, A2, and DTNB light chains from ordinary muscle myosin resembled one another in ultraviolet absorption spectrum, as did D1 and D2 light chains from dark muscle myosin. However, the absorption spectra of the former three differed from those of the latter two.
2. Amino acid compositions of A1 and A2 light chains resembled each other, except for a few amino acids such as lysine, proline, and alanine. Tryptophan was detected only in DTNB light chain. D1 and D2 light chains showed general similarity, except for a remarkably higher proline content in D1.
3. Anti-Al (or anti-A2) antiserum exhibited a cross-reaction against A2 (or A1) in both immunoelectrophoresis and ELISA, indicating an immunochemical similarity of these two alkali light chains.
4. No precipitin line appeared when anti-A1 or anti-A2 antiserum was diffused against DTNB light chain in immunoelectrophoresis. In ELISA, however, each pair showed cross-reactivity values as high as 50-80%, values which were rather higher than those obtained with heterologous alkali light chains (10-40%). Anti-DTNB light chain antiserum reacted with either alkali light chain in both methods.
5. Anti-DI antiserum cross-reacted against D2, and anti-D2 antiserum did against D1.
6. These myosin light chains exhibited a high immunochemical tissue-specificity.View full abstractDownload PDF (1675K) -
Noriaki SHIMIZU, Yoshihiro SOKAWA1983 Volume 94 Issue 5 Pages 1421-1428
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA high level of activity of (2'-5') oligoadenylate synthetase (2-5A synthetase) was detected in pig spleens not treated with exogenous interferon. The enzyme recovered from homogenates of pig spleens was partially purified by the use of a combination of ion exchange gels (DEAE-Sephadex and CM-Sepharose) and affinity gels (2', 5'-ADP-Sepharose and poly (I): poly (C)-agarose). The specific activity of the final sample was 32, 800 nmol AMP polymerized/h/mg protein at 33°C, and the enzyme was able to convert over 80% of ATP into 2-5A after a 24-h incubation; penta-adenylate was the major product (41% of total product). The 2-5A synthetase obtained was eluted from Sephacryl S-200 at around the position of a protein with Mr=100, 000.
By using the purified 2-5A synthetase from pig spleens as an antigen, rabbit antiserum to this enzyme was prepared. The antibody bound to protein A-Sepharose absorbed the activity of 2-5A synthetase induced by interferon in pig cells. This result shows that the IFN-induced 2-5A synthetase shares antigenic determinants with the synthetase in spleens. Neither human nor mouse 2-5A synthetase combined with the antibody to porcine 2-5A synthetase. Thus, the antigenic structure of this enzyme is different from species to species.View full abstractDownload PDF (642K) -
Kazufumi KURODA, Ritsuko KAGIYAMA1983 Volume 94 Issue 5 Pages 1429-1441
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe immunological and the biochemical relationships between F-type pyocins and phage KFI, which is cross-reactive with anti-F-type pyocin sera, were studied. The primary structures of six subunit proteins of each pyocin were also compared among three F-type pyocins, pyocin Fl, F2, and F3.
Both anti-pyocin F1 and anti-pyocin F3 sera gave precipitin bands with phage KF1 by Ouchterlony's test, and were found to bind to minor subunit proteins P3 and P6 of the phage, which were separated by SDS-polyacrylamide gel electrophoresis. Electron microscopic studies showed that these sera bound to some loci of the rod part of the phage tail. These results showed that the subunit proteins P3 and P6 carried antigenically common parts to F-type pyocin and that P3 and/or P6 were components of the rod part of the phage KF1. The subunit proteins P3 and P6 of the phage were of the same mobilities in SDS-polyacrylamide gel electrophoresis as those of band 1 and band 5 of F-type pyocins, respectively. Tryptic peptide mapping after iodination with 125I also showed a partial homology between P3 and band 1, and between P6 and band 5.
The tryptic peptide mapping of the six subunit proteins of each F-type pyocin showed that the primary structure of subunit protein band 1 was almost the same among these pyocins, and subunit protein bands 2, 3, 5, and 6 showed were similar. Only subunit protein band 4 was different among these pyocins. The difference in the action spectra of pyocin F1, F2, and F3 is probably due to the difference in the primary structure of subunit protein band 4, which is a component of the fiber part.View full abstractDownload PDF (4797K) -
Yutaka YOSHIDA, Kenji SAKIMURA, Tooru MASUDA, Etsuko KUSHIYA, Yasuo TA ...1983 Volume 94 Issue 5 Pages 1443-1450
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeuron-specific enolase (NSE), and non-neuronal enolase (NNE) which exists in many tissues including liver but is localized in glial cells within the nervous system, were synthesized in the rabbit reticulocyte cell-free translation system programed with brain mRNAs. The in vitro synthesized NSE and NNE were indistinguishable from the two enzymes purified from rat brains. NSE mRNA activity was found only in brain RNAs, while NNE mRNA activity existed in brain RNAs as well as liver RNAs. In developing brains, the level of translatable NSE mRNA was low at the embryonic stage and at birth, increased rapidly from about 10 days postnatal, and reached the adult level, while that of NNE mRNA was high at the embryonic stage and at birth, followed by a slight decrease then a gradual rise to adult levels. These changes correlated with the developmentally regulated appearance and accumulation pattern of each of the two enzymes. These results suggest that the levels of NSE and NNE are controlled primarily by the level of each of the two translatable mRNAs. In developing livers, only the NNE mRNA activity was detected and its level generally paralleled the changes in the level of NNE.View full abstractDownload PDF (2731K) -
Katsura INOUE, Toshiharu HASE, Peter BÖGER1983 Volume 94 Issue 5 Pages 1451-1455
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of a [2Fe-2S] ferredoxin isolated from Bumilleriopsis filiformis, a yellow-green alga, was determined by using conventional techniques. It consisted of 98 amino acid residues with a microheterogeneity at the amino-terminus: Ala/Glu-Thr-Tyr-Ser-Val-Thr-Leu-Val-Asn-Glu-Glu-Lys-Asn-Ile-Asn-Ala-Val-Ile-Lys-Cys-Pro-Asp-Asp-Gln-Phe-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Gln-Gly-Ile-Glu-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala- Cys-Ser-Thr-Cys-Ala-Gly-Lys-Val-Leu-Ser-Gly-Thr- Ile-Asp-Gln-Ser-Glu-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Met-Gly-Ala-Gly-Phe-Leu-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Lys-Val-Gln-Thr-His-Ala-Glu-Asp-Asp-Leu-Tyr. No prominent structural feature was noted in this ferredoxin in comparison with other homologous ferredoxins. From the structural comparison, B. filiformis was placed taxonomically close to filamentous blue-green algae and red algae.View full abstractDownload PDF (329K) -
Toshiharu HASE, Katsura INOUE, Naoko HAGIHARA, Hiroshi MATSUBARA, Myrt ...1983 Volume 94 Issue 5 Pages 1457-1464
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of a ferredoxin from a unicellular blue-green alga, Aphanothece halophitica, was established by the conventional methods. Total number of residues was 98 lacking only tryptophan. A most probable phylogenetic tree was constructed for 19 algal ferredoxins on the basis of an amino acid difference matrix made from the sequence comparison. A. halophitica has been classified as a unicellular blue-green alga in the same genus to which Aphanothece sacrum belongs, but the tree indicates A. halophitica ferredoxin to be very close to those of the members of filamentous blue-green algae. The tree divides prokaryotic and eukaryotic algal ferredoxins into several groups, suggesting that the ferredoxin phylogenetic tree reflects the evolutionary trails of various algae, which is also reflected in the structural characteristics, particularly in the presence of gaps. Other notable features are presented in considering algal taxonomy.View full abstractDownload PDF (506K) -
Kaname KAWAJIRI, Kazuhiro SOGAWA, Osamu GOTOH, Yusaku TAGASHIRA, Masam ...1983 Volume 94 Issue 5 Pages 1465-1473
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly (A)+ RNA was isolated from membrane-bound polysomes of the livers of 3-methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte lysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5 of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli x 1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56, 000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MG, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridization with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. Acad. Sci. U. S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner. The former cDNA strongly hybridized with MC- and PCB-induced mRNA, while the latter one did so with PB- and PCB-induced mRNA. The mRNA hybridized with P-450MC cDNA showed a single band with almost the same mobility as observed with the mRNA hybridized with P-450b cDNA, indicating that the mRNA for P-450MC is approximately 2, 000 bases long, as is the case with the P-450b mRNA. From these results, we conclude that the isolated cDNA clones carry a complementary sequence to the cytochrome P-450MC mRNA.
A preliminary sequence analysis of the longer cDNA insert (P-34) revealed that the longer cDNA insert indeed contained the coding nucleotide sequence for the reported NH2-terminal sequence of 30 amino acids of one of the two MC-inducible cytochrome P-450's, P-450d (Botelho et al. (1982) Biochemistry 21, 1152-1155) (data to be published elsewhere).View full abstractDownload PDF (2797K) -
Toshihiro YASUDA, Yasuo INOUE1983 Volume 94 Issue 5 Pages 1475-1481
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of a series of adenosine-3'-alkylphosphates, Ap(CH2)n-1CH3 with n=1-8, on the binding and catalytic activities of RNase T2 were investigated. The inhibition of the RNase T2-catalyzed reaction by a series of adenosine-5'-alkylphosphates, CH3(CH2)n-1pA with n=1-7, was also studied. Multiple regression analyses of the observed kinetic constants were carried out to determine the contribution of polar and hydrophobic effects to kcat, kcat/Kmi, and cosubstrate and inhibitor bindings (Kmi and K1). The data show that the pKmi for Ap(CH2)n-1CH3 increases uniformly with n up to n=4, then remains constant. By contrast pKj for CH3(CH2)n-1pA is independent of n. The data for log kcat correlated well with polar and hydrophobic variables. On the other hand, log kcat/Kmi is independent of a hydrophobic effect, and the data were fitted by a single polar variable equation. The contribution of hydrophobic regions at or near the active site of RNase T2 is discussed. The present results also offer evidence for the existence of an isomerization step of the first formed enzyme-substrate complex.View full abstractDownload PDF (512K) -
Tadayoshi UEZATO, Michiya FUJITA1983 Volume 94 Issue 5 Pages 1483-1488
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPostnatal change of rat intestinal alkaline phosphatase from suckling to adult type occurred at the weaning stage (Uezato et al. (1981) Biochem. Int. 2, 561). Two distinct forms were observed in suckling rat small intestine. One was soluble and the other was membrane-bound. The ratio of the soluble to the membrane-bound form increased after birth until these forms were replaced by the adult type, which consisted of membrane-bound forms different from that of sucklings. The soluble form was demonstrated almost exclusively during the suckling period and was distributed in the distal third of the small intestine. The postnatal increase of alkaline phosphatase was enhanced by administration of hormones such as cortisone and thyroxine. On the other hand, acid β-galactosidase activity in the distal segment was reduced rapidly by the same treatment. The change of alkaline phosphatase from suckling to adult type was accelerated by the injected hormones. In the distal segment the ratio of soluble to membrane-bound activity in the homogenate prematurely decreased after administration of the hormones. Between 16 and 18 days of age, alkaline phosphatase of hormone-treated rat small intestine changed completely from fetal-suckling type to typical adult type in both the proximal and distal segments.View full abstractDownload PDF (1593K) -
Yurie KUREHA, Yoshikatu ETO1983 Volume 94 Issue 5 Pages 1489-1492
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArylsulfatases B1 and B2 isolated from human brain were introduced into neonatal type MSD fibroblast culture. Arylsulfatase B2 was linearly taken up into MSD fibroblasts with respect to time and the dose of enzyme, whereas arylsulfatase B1 enzyme was practically not taken up into cells. Uptake of arylsulfatase B2 was inhibited in the presence of mannose 6-phosphate. These data suggest that the biological functional difference between arylsulfatases B1 and B2 is due to the presence of a phosphorylated residue acting as a recognition marker on one of the enzymes.View full abstractDownload PDF (249K) -
Mayumi ONO, Miho ANDO, Tatsuo SHIMADA, Koji FURUNO, Keitaro KATO, Mich ...1983 Volume 94 Issue 5 Pages 1493-1503
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4CI, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 μg/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, β-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 μg/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 μg/ml of chloroquine.View full abstractDownload PDF (2303K) -
Nuran NABI, Toyoko ISHIKAWA, Masao OHASHI, Tsuneo OMURA1983 Volume 94 Issue 5 Pages 1505-1515
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytoplasmic free and membrane-bound ribosomes were isolated from bovine adrenal cortex, and characterized. Contributions of free and bound ribosomes to the synthesis of NADPH-adrenodoxin reductase (AdR) and adrenodoxin (Ad) were determined by examining the presence of their nascent peptides on isolated ribosomes. Nascent peptides were released from the ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of AdR and Ad were separately isolated by immunoprecipitation using antibodies. AdR nascent peptides were associated with free and loosely-bound ribosomes, whereas Ad nascent peptides were associated with free, loosely-bound and tightly-bound ribosomes. Smaller nascent peptides of AdR were carried by free ribosomes, whereas larger nascent peptides were preferentially carried by loosely-bound ribosomes. In the case of Ad, smaller nascent peptides were more abundant in free ribosomes than in bound ribosomes. The nascent peptides of Ad were released from bound ribosomes of rough microsomes to the aqueous milieu by puromycin treatment, suggesting the release of completed Ad peptides into the cytoplasm in cells.View full abstractDownload PDF (2103K) -
Nuran NABI, Shiro KOMINAMI, Shigeki TAKEMORI, Tsuneo OMURA1983 Volume 94 Issue 5 Pages 1517-1527
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit antibodies against cytochrome P-450 (SCC), P-450 (11β), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11β), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11β), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11β) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.View full abstractDownload PDF (1988K) -
Nuran NABI, Tsuneo OMURA1983 Volume 94 Issue 5 Pages 1529-1538
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytoplasmic free and bound polysomes isolated from bovine adrenal cortex were used to program protein synthesis in rat liver cell sap (175, 000×g soluble material) and wheat germ lysate S-175 (175, 000×g soluble material) systems. Both free and bound polysomes were translated with high fidelity and equal efficiency in the cellfree systems. Synthesis of adrenodoxin reductase (AdR) and adrenodoxin (Ad) in the cell-free systems was determined by the immunoprecipitation of the translation products using monospecific antibodies, and we found that AdR was synthesized by free polysomes whereas Ad was synthesized by both free and bound polysomes as a large precursor (pAd) having a molecular weight of approximately 22, 000 daltons, which is about 10, 000 daltons larger than mature Ad. The existence of a large precursor of Ad was also confirmed by its synthesis in a cell-free system programmed by total RNA isolated from bovine adrenal cortex. Bovine adrenal cortex mitochondria imported in vitro-synthesized AdR and pAd to the inside of the organelle in the absence of protein synthesis in a cell-free system, and processed pAd to the mature form during the import. These results suggest that cytoplasmically synthesized AdR and Ad are translocated posttranslationally into the matrix of mitochondria in adrenal cortex cells.View full abstractDownload PDF (805K) -
Sugie HIGASHI-FUJIME1983 Volume 94 Issue 5 Pages 1539-1545
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn vitro movement of fibrils composed of actin and myosin filaments purified from skeletal muscle was observed by dark field microscopy during superprecipitation at low ionic strengths at room temperature. The movement was activated by phosphorylation of light chain (LC2) of myosin. The activity of the movement was evaluated in terms of the spreading of the area where the fibrils were moving. Adenosine triphosphatase activity of actomyosin was also enhanced by phosphorylation of LC2 and was correlated with the activity of the in vitro movement.View full abstractDownload PDF (1660K) -
Minoru TAMURA, Toshitsugu YUBISUI, Masazumi TAKESHITA1983 Volume 94 Issue 5 Pages 1547-1555
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1. 6. 2. 2] was solubilized by digestion with lysosomes, and purified 8, 500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31, 000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS).
Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor).
The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 μM, respectively. The apparent Km value for cyt. b5 was 14.3 μM in 10mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1, 190 pmol cyt. b5 reduced/min/mg of protein.
The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type.
These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue).
An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.View full abstractDownload PDF (2140K) -
Takashi KAGEYAMA, Akihiko MORIYAMA, Kenji TAKAHASHI1983 Volume 94 Issue 5 Pages 1557-1567
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive pepsinogens were purified to homogeneity from the gastric mucosa of Asiatic black bear and termed pepsinogens I-1, I-2, II-1, II-2, and III. Pepsinogen II-1 was the major component and accounted for more than half of the total pepsinogens. Their molecular weights were estimated to be 40, 000 for pepsinogens I-1 and I-2, 38, 000 for pepsinogens II-1 and II-2, and 42, 000 for pepsinogen III. They resembled each other in amino acid composition, except that pepsinogens I-1 and I-2 contained larger numbers of basic residues than the others. Pepsinogen III was a glycoprotein containing about 3.7% carbohydrate. Each was activated to the corresponding pepsin and their enzymatic characteristics were investigated. The optimal pH against hemoglobin was about 2.2 for pepsin I-1, and about 2.5 for pepsins II-1, II-2, and III. Each pepsin was inhibited by pepstatin as well as porcine pepsin and also by diazoacetyl-DL-norleucine methyl ester, 1, 2-epoxy-3-(p-nitrophenoxy)-propane, and p-bromophenacyl bromide. Each pepsin could hydrolyze N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine, but the specific activity was much lower than that of porcine pepsin. Activation peptides corresponding to residues 1-43, 1-25, and 26-43 were isolated from an activation mixture of pepsinogen II-1. The amino acid sequences of these peptides and of the NH2-terminal portions of pepsinogen II-1 and pepsin II-1 were determined, resulting in the complete NH2 terminal 60-residue sequence of pepsinogen II-1.View full abstractDownload PDF (1232K) -
Kuniji TANAKA, Takashi KARIGANE, Fumio YAMAGUCHI, Shigeko NISHIKAWA, N ...1983 Volume 94 Issue 5 Pages 1569-1578
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLevan fructotransferase of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2, 6':6, 2' dianhydride (difructose anhydride IV) from levan by an intramolecular transfructosylation reaction, was purified to give a single protein band of pI 4.5-4.7 on isoelectric focusing. It had a molecular weight of 128, 000 on gel-filtration on Sephadex G-200 and 60, 000 on SDS-polyacrylamide disc gel-electrophoresis, suggesting that the enzyme is composed of two identical subunits. The shortest levanoligosaccharide chain required for the difructose anhydride IV formation was determined to be tetraose. TLC of the enzymic digest of a modified levanhexaose derived from levanhexaose by the reduction of the reducing end to an alditol residue with sodium borohydride gave the difructose anhydride IV spot, suggesting that the enzyme attacks the modified levanhexaose molecule from the direction of the non-reducing fructose end. The enzymic digests of levantetraose, pentaose, and-hexaose as the substrate gave, in addition to the difructose anhydride IV spot, spots of oligofructans of lower mobility than the original substrate on TLC. From the digest of levantetraose, a hexaoligofructan and a smaller amount of a pentaoligofructan but no fructose were separated, indicating enzymic intermolecular levanbiosyl and fructosyl transfer reactions.View full abstractDownload PDF (1508K) -
Toshiko SHIBAYAMA, Kazuyasu NAKAYA, Yasuharu NAKAMURA1983 Volume 94 Issue 5 Pages 1579-1586
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe compared the phosphorylation of nucleolar proteins during the cell cycle of Physarum polycephalum labeled by pulse and continuous labeling methods in vivo with that obtained by in vitro labeling of isolated nucleoli. Both the phosphorylating activity of nucleoli and total incorporation of radioactive phosphate into nucleolar proteins increased and reached a maximum about 1.5-2.0 h before mitosis, confirming our previous observation. Analyses of labeled nucleolar proteins by SDS-polyacrylamide gel electrophoresis and by autoradiography indicated that most of the phosphoproteins labeled by in vitro labeling were labeled by in vivo pulse labeling. At least 10 nucleolar proteins underwent phosphorylation, which closely followed the cell cycle-dependent changes of the total phosphate incorporation into the nucleolar proteins. When mitosis was delayed by UV-irradiation, the maximal incorporation of radioactive phosphate into nucleolar proteins in vivo was not observed at the usual time, it shifted to about 2 h before the delayed mitosis, and the same set of nucleolar proteins that were phosphorylated without UV-irradiation were most heavily phosphorylated at this time. These results suggest the possibility that the increased phosphorylation of nucleolar proteins of Physarunz just before mitosis is related to the onset of subsequent mitosis.View full abstractDownload PDF (2094K) -
Ken-ichiro TAKAMIYA1983 Volume 94 Issue 5 Pages 1587-1593
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN, N'-Dicyclohexylcarbodiimide (DCCD) inhibited dark re-reduction of cytochrome c2 and reduction of b-type cytochrome, both of which are closely associated with electron transfer involving a cytochrome b-c2 oxidoreductase, after a single-turnover flash excitation in the chromatophore membranes from a photosynthetic bacterium, Rhodopseudomonas sphaeroides. Rapid proton uptakes (HI+, HII+) and the formation of the membrane potential registered by carotenoid bandshift phase III were also inhibited by DCCD. The electron transfer was inhibited in the presence of either valinomycin or carbonylcyanide-m-chlorophenylhydrazone (CCCP). These results indicated that DCCD inhibited the electron transfer involving the cytochrome b-c2 oxidoreductase in the bacterium. The inhibition was irreversible.
A hydrophilic carbodiimide, I-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), did not affect the above-mentioned reactions. Thus, DCCD may interact with the hydrophobic region (s) in the chromatophore membranes from photosynthetic bacteria resulting in the inhibition (s) of the photosynthetic cyclic electron transfer.View full abstractDownload PDF (460K) -
Kentaro SHIMURA, Masato OKADA, Hiroshi SHIRAKI, Hachiro NAKAGAWA1983 Volume 94 Issue 5 Pages 1595-1603
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInhibition of conversion from IMP to uric acid, which interferes with both spectrophotometric and radioisotopic assays of IMP dehydrogenase, by addition of allopurinol (0.1mM), an inhibitor of xanthine oxidase, to the incubation system made it possible to determine the enzyme activity in crude liver extracts. With this improved assay method, the regulatory properties of the enzyme in crude extracts of liver and Yoshida sarcoma ascites cells were examined. In both tissues IMP dehydrogenase was found in the postmicrosomal supernatant. However, further centrifugation resulted in precipitation of the enzyme, the enzyme from Yoshida sarcoma ascites cells being precipitated more easily than that from rat liver. It was also found that IMP dehydrogenase activity increased during liver regeneration and that this increase was associated with the precipitate from the postmicrosomal fraction. These findings suggest that such a large sedimentable complex including IMP dehydrogenase might be formed in relation to cell growth.
Most of the enzyme activity in rat liver and Yoshida sarcoma ascites cells was extracted in the supernatant obtained by centrifugation at 105, 000×g for 4 h after treatment of tissue homogenates with 1M KCl, 0.75M (NH4)2SO4, 2M dimethylsulfoxide, 2 M KSCN, 25% glycerol, or 0.8 M guanidine-HCI. Treatment with 2 deoxycholate, 2% Triton X-100 or 2M urea gave limited extraction. The enzyme was retained on a phenyl-Sepharose CL-6B or octyl-Sepharose CL-6B column and eluted with 0.8M guanidine-HCl. These results suggested that the enzyme molecule has not only ionic but also hydrophobic domains, through which it interacts with other molecules of the enzyme itself and/or postmicrosomal cellular components.View full abstractDownload PDF (653K) -
II. Purification and Properties of the Enzyme from Yoshida Sarcoma Ascites Tumor CellsMasato OKADA, Kentaro SHIMURA, Hiroshi SHIRAKI, Hachiro NAKAGAWA1983 Volume 94 Issue 5 Pages 1605-1613
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1. 2. 1. 14] tended to form a precipitable complex (es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68 K daltons. Amino acid analyses indicated a subunit molecular weight of 68, 042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127 K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200 K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics.
The Km values for IMP and NAD were calculated to be 12 and 25 μM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 μM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. CI-IMP also inactivated the enzyme and IMP prevented this inactivation. These findings suggest that IMP binds to a thiol group of the enzyme.
IMP dehydrogenases from Yoshida sarcoma ascites cells and rat liver are immunologically identical.View full abstractDownload PDF (1709K) -
Genichiro OSHIMA1983 Volume 94 Issue 5 Pages 1615-1620
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEgg white lysozyme was rapidly and extensively hydrolyzed by chymotrypsin in the presence of negatively charged phospholipid vesicles. The extent of hydrolysis of lysozyme by chymotrypsin depended on the amount of phospholipid present. The optimum amount of phospholipid varied with the amounts of both lysozyme and chymotrypsin. The proteolysis was strongly inhibited at high ionic strength. The amidolytic activity of chymotrypsin against a synthetic substrate was inhibited by phospholipid.
Purified phosphatidic acid and phosphatidylethanolamine from egg yolk induced susceptibility of lysozyme to chymotrypsin, whereas synthetic dimyristoyl phosphatidylcholine did not. The extent of the hydrolysis was smaller with phosphatidic acid and phosphatidylethanolamine than with phospholipid mixture, indicating that vesicles of phospholipid mixture were more effective than those of phosphatidic acid or phosphatidylethanolamine in enhancing the proteolysis of lysozyme by chymotrypsin.View full abstractDownload PDF (482K) -
Masaru TANOKURA1983 Volume 94 Issue 5 Pages 1621-1630
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 270 MHz 1H NMR spectra of 3'-UMP and 3'-CMP were observed in the presence of a two-fold molar excess of bovine pancreatic RNase A [EC 3. 1. 27. 5] at various pHs. For the C (5), C (6), and C (1') protons of these nucleotides, the pH profiles of chemical shifts induced by binding to RNase A were obtained by plotting the differences between chemical shifts in the presence and the absence of RNase A against pH. Such profiles were bell-shaped for the C (5) and C (6) protons of both 3'-UMP and 3'-CMP. However the profiles of C (1') protons were not bellshaped but appeared to consist of two bell-shaped curves and reflect the dissociations of at least three ionizable groups. The observations for the C (1') protons suggest that there are at least two forms of complexes different from each other in the interaction reflecting the chemical shift of the C (1') proton. In order to clarify the interacting sites of ribonucleotides affecting the induced shift profile of the C (1') proton, the pH titration curves were observed for 3'-dCMP in the presence of RNase A. The induced shift profile was bell-shaped for the C (1') proton as well as for the C (5) proton of the base. This indicates that the interaction at the O (2') H [or O (2')] sites of ribonucleotides causes the two forms of complexes of 3'-UMP and 3'-CMP with RNase A. The interacting sites and modes were discussed with these and the pH titration curves of His-12, His-119, and Phe-120 of RNase A in the presence of a three-fold molar excess of ribonucleotides.View full abstractDownload PDF (688K) -
Shoichi ISHIURA, Ikuya NONAKA, Takehisa FUJITA, Hideo SUGITA1983 Volume 94 Issue 5 Pages 1631-1636
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have examined the effect of cycloheximide on bupivacaine-induced, macrophage-mediated muscle degeneration in rats and obtained the following results.
1. Direct intramuscular injection of bupivacaine into soleus muscle caused uniform muscle degeneration (Ishiura, S. et al. (1983) J. Biochem. 94, 311-314). The degeneration was most prominent 48 h after injection, with many infiltrating macrophages. Intraperitoneal administration of cycloheximide prevented the bupivacaine-induced macrophage invasion and decrease of structural proteins 48 h after injection.
2. Cycloheximide inhibited the increase in lysosomal enzymes in bupivacaine-treated muscle.
3. The peritoneal macrophages increased in number immediately after bupivacaine injection, while cycloheximide inhibited their accumulation. These results indicated that cycloheximide inhibited muscle degradation by preventing macrophage proliferation.View full abstractDownload PDF (1175K) -
Tomohiro MEGA, Yoshio MATSUSHIMA1983 Volume 94 Issue 5 Pages 1637-1647
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral β-D-glucopyranosides (p-nitrophenyl, phenyl, and ethyl), 1-thin-β-D-gluco-pyranosides, and phenyl 2-deoxy, 3-deoxy, 4-deoxy, and 6-deoxy β-D-glucopyrano-sides were synthesized and used to study the mechanism of the enzymatic action of Taka-β-glucosidase [EC 3. 2. 1. 21 Aspergillus oryzae]. Kinetic constants of the enzyme for these glycosides were determined from S/V-S or 1/V-1/S plots, and the hydrolysis rates of these compounds with the enzyme, acid (3 N HCl) and alkali (3 N NaOH) were compared.
Inhibition of the enzyme by 1, 5-anhydroglucitol, glucal, dihydroglucal, and 1, 6-anhydroglucopyranose was also examined. Glucal and 1, 5-anhydroglucitol showed strong competitive inhibition. Free energy of binding of each hydroxyl group of glucosidic glucose with the enzyme was estimated from Kms of phenyl β-glucoside and its deoxy analogues, and also K1 values of some inhibitors. The free energies of binding of 2-OH, 3-OH, 4-OH, and 6-OH were calculated to be 1.1, 2.4, 0.7, and 1.8 kcal/mol, respectively. The free energy of binding of phenoxide at C-1 (0.3 kcal/mol) was calculated from the Km of Ph-β-Glc and K1 of 1, 5-anhydroglucitol. The energy of binding of 5-CH2OH (2.3 kcal/mol) was obtained from the Km of Ph-β-Glc and that of Ph-β-Xyl. The sum (6.8 kcal/mol) of each partial binding free energy was close to the value of binding free energy of Ph-β-Glc (7.0 kcal/mol) calculated by the equation; -ΔGbind=- RT In Km-TΔGSmix, showing that the methods of estimation of each binding energy used in the present study seemed reasonable.
Glucal, having a pyranose form distorted slightly, showed strong competitive inhibition and the K1 of this inhibitor was smaller than the Km of Ph-β-Glc, suggesting that the sugar ring bound to the active site was distored to a half chair form which is labile to acid hydrolysis.
Kinetic data, especially Kcat, showed that hydroxyls of C-2, C-3, and C-4 played important roles not only in the binding of the substrate to the active site, but also in the increase of the hydrolysis rate.View full abstractDownload PDF (852K) -
Francisco MUÑOZ, Matias A. VALLES, Josefa DONOSO, Gerardo ECHEV ...1983 Volume 94 Issue 5 Pages 1649-1659
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe association equilibrium of phosphorylase b, induced by AMP and in the presence of Mg2+, has been shown to be a reversible process that follows second order and first order reversible rate laws in the direction of tetramerization and dimerization respectively, this fact being independent of temperature and of enzyme and AMP concentrations. Moreover, rate and equilibrium constants have been evaluated and their dependence on temperature and AMP concentration studied in this work.
An important role that the existence of two classes of AMP binding sites per enzymatic subunit plays in the aggregation properties of the enzyme has also been emphasized. In the presence of 0.1 and 1mM AMP (binding to the high affinity site), the values of the change in enthalpy, activation energy of dimerization and activation energy of tetramerization are: -36 kcal/mol, +36 kcal/mol, and 0 kcal/mol respectively. Binding of AMP to the low affinity site (10mM AMP) yields significant changes in the self-association equilibrium, since the preceding parameters reach the following values: -18, +32, and +14 kcal/mol.View full abstractDownload PDF (744K) -
Fumio SAKIYAMA, Yasushi KAWATA1983 Volume 94 Issue 5 Pages 1661-1669
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1H and 13C NMR titrations were performed on PMS-St. trypsin derived by PMSF modification of Ser 195 in Streptomyces erythraeus trypsin, which is devoid of autocatalytic degradation activity at pH_??_8. NMR titration of the imidazole C2 proton showed that His 57 had the pKa, value of 6.9 in both native St. trypsin and PMS-St. trypsin, suggesting that the adjacent hydroxyl group of Ser 195 had no effect or a very weak effect on the acid-base properties of the imidazole ring in the catalytic triad.
A small change in the chemical shift of the isotopically enriched methylene carbon in the PMS moiety of [methylene-13C] PMS-St. trypsin was observed between pH 6 and 8. The titration curve had an inflexion at pH 6.9 and the mode of transition was apparently sigmoidal, although a Hill coefficient of more than unity was suggested. It is thus likely that His 57 is responsible for this transition.
Based on these results, the role of His 57 in the catalytic triad of the active site in serine protease is discussed.View full abstractDownload PDF (654K) -
Eisuke NISHIDA, Yasutaka OHTA, Hikoichi SAKAI1983 Volume 94 Issue 5 Pages 1671-1683
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe action of the 88 K protein/actin complex (88 K/A) and cytochalasin B on various aspects of actin polymerization kinetics was investigated, and the results were interpreted in terms of the condensation polymerization and treadmilling mechanism for actin polymerization. A substoichiometric concentration of 88 K/A promotes actin nucleation under physiological salt conditions, especially in the presence of Ca2+. In addition, it reduces both the elongation rate and the depolymerization rate by up to 70% and inhibits annealing of the actin filaments. As a consequence, the average length of actin filaments polymerized with 88 K/A becomes less than that of a control. These data indicate that 88 K/A caps one end of actin filaments or actin oligomers where in the absence of 88 K/A the rates for both association and dissociation of monomers are faster than at the other end. In a KCl/MgCl2 medium, 88 K/A increases the steady state monomer concentration (the critical concentration) to a limited extent. This is explained by assuming that 88 K/A caps the lengthening end (in treadmilling) of actin filaments, where the critical concentration is lower than at the other end (the shortening end). Moreover, cytochalasin B which has been shown to bind to the barbed end of actin filaments does not affect the 88 K/A-nucleated actin polymerization. Therefore, it is strongly suggested that 88 K/A caps the barbed end of actin filaments and that the barbed end is the lengthening end as well as the rapidly growing and rapidly depolymerizing end. The result obtained in the study on the action of cytochalasin B was consistent with this suggestion.View full abstractDownload PDF (2915K) -
Kazuo ASAOKA, Kenji TAKAHASHI1983 Volume 94 Issue 5 Pages 1685-1688
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConditions have been examined for the use of o-dinitrobenzene as a substrate for colorimetric assay of glutathione S-transferases. Activities can be determined by measuring nitrite released enzymatically from the substrate using a diazo-coupling method with N- (1-naphthyl) ethylenediamine dihydrochloride and sulfanilamide. The assay can be done in the presence of large amounts of reduced glutathione (GSH), cysteine, and protein, and is capable of quantitating the enzyme activity in about 30 μg (wet weight) of monkey liver, corresponding to 0.1 μg of purified glutathione S-transferase from the same source. This method is suitable for assaying simultaneously a large number of samples with reasonable sensitivity and speed.View full abstractDownload PDF (274K) -
Hiroshi NAKADA, Yutaka TASHIRO1983 Volume 94 Issue 5 Pages 1689-1695
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding proteins for asialoorosomucoid were prepared from rat liver previously labeled in vivo with [3H] leucine by affinity chromatography on asialoorosomucoid-Sepharose 4B. They were subjected again to the same affinity chromatography and eluted into two fractions successively with 10mM Tris-HCl buffer, pH 7.8, containing 1.25M NaCl, 1% Triton X-100 and 50mM lactose and 20mM ammonium acetate buffer, pH 6.0, containing 1.25M NaCl and 1% Triton X-100, and designated as ABP-I and ABP-II (asialoorosomucoid binding proteins), respectively. ABP-I corresponds to the receptor protein specific for asialoglycoproteins which has been extensively investigated by Ashwell and collaborators (J. Biol. Chem. 254, 1038-1043, 1979).
ABP-II is different from ABP-I in several properties such as molecular weight, antigenicity and solubility. The molecular weight of ABP-II was estimated to be 29, 000 by SDS-PAGE. On gel filtration it behaved as a pentamer with an apparent molecular weight of 150, 000. Unlike ABP-I, ABP-II showed no detectable binding activity when assayed according to the procedures of Hudgin et al. (J. Biol. Chem. 249, 5536-5543, 1974). The calcium ion was, however, essential for the binding of ABP-II to asialoorosomucoid-Sepharose 4B similar to ABP-I. ABP-II can be extracted from the total microsomes of rat liver in 1.0M NaCl by sonication after freezing and thawing. This suggests that ABP-II is either a soluble protein or a peripheral membrane protein loosely attached to the intracisternal cavities of the microsomal membranes.View full abstractDownload PDF (1514K) -
Shingo NIIMI, Toshikazu NAKAMURA, Katsuhiko NAWA, Akira ICHIHARA1983 Volume 94 Issue 5 Pages 1697-1706
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTryptophan 2, 3-dioxygenase [EC 1.13.11.11] in primary cultures of adult rat hepatocytes was induced 3-4 fold by 1 μm dexamethasone and 6-7 fold by dexamethasone plus glucagon (0.1 μM). Changes of the enzyme activity, amount of enzyme, measured by immunotitration, and rate of enzyme synthesis, assayed by measurement of [3H] leucine incorporation into the enzyme protein, were closely correlated. Furthermore, in a reticulocyte lysate system for cell-free protein synthesis, mRNA of the enzyme was translated to the protein corresponding to the subunit of tryptophan 2, 3-dioxygenase, which was identified by SDS-polyacrylamide gel electrophoresis. The activity of translatable mRNA of the enzyme was increased more than 10-fold by dexamethasone and its final content in total mRNA was 0.34%. Glucagon alone did not increase mRNA activity, but dexamethasone plus glucagon increased mRNA activity to twice that with dexamethasone alone, the maximal content of the mRNA being 0.77% of the total mRNA content 12 h after addition of hormones.
Insulin (0.1 μM) caused 75% inhibition of the maximum increase of mRNA activity of the enzyme induced by dexamethasone and glucagon. Epinephrine (10 μM) also caused 58% inhibition of the maximum increase. Insulin and epinephrine also suppressed increase of mRNA of tryptophan 2, 3-dioxygenase induced by dexamethasone alone. Therefore, dexamethasone alone or together with glucagon stimulated transcription of tryptophan 2, 3-dioxygenase increasing its mRNA and enzyme synthesis in hepatocytes. Conversely, insulin and epinephrine suppressed these increases of mRNA synthesis and thus decreased enzyme synthesis.View full abstractDownload PDF (738K) -
Fujiko SUZUKI, Kanefusa KATO, Takashi NAKAJIMA1983 Volume 94 Issue 5 Pages 1707-1710
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen rat epididymal fat-pad pieces were incubated in vitro with 10 μm epinephrine, S-100 protein in the tissue was markedly decreased by release into the medium. The release of adipose S-100 protein was also enhanced by norepinephrine (10 μm), isoproterenol (10 μm), and dibutyryl cyclic AMP (5mM), but not by insulin (0.8 μm). The enhancement of S-100 protein release by 10 μM epinephrine was completely inhibited by 10 μM propranolol. These results suggest that the release of adipose S-100 protein is regulated by the β3-adrenergic effect of catecholamines.View full abstractDownload PDF (252K) -
Tadashi OGISHIMA, Yoshiie OKADA, Shiro KOMINAMI, Shigeki TAKEMORI, Tsu ...1983 Volume 94 Issue 5 Pages 1711-1714
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPartial amino acid sequences of two mitochondrial cytochrome P-450's, P-450 (SCC) and P-450 (11β), and two microsomal cytochrome P-450's, P-450 (C-21), and P-450 (17α, Lyase), from adrenal cortex were analyzed and compared. Mitochondrial P-450's and microsomal P-450's were different in the amino acid sequences at their NH2-terminals. The sequences of microsomal P-450's started from terminal methionine and were highly hydrophobic, whereas those of mitochondrial P-450's lacked NH2-terminal methionine and were not hydrophobic. These findings strongly suggest that the NH2-terminal portions of newly synthesized P-450's determine their intracellular localization to different cell organelles.View full abstractDownload PDF (335K) -
Atsushi HARA, Tamotsu TAKETOMI1983 Volume 94 Issue 5 Pages 1715-1718
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSphingosylphosphorylcholine prepared from native sphingomyelin by the Kaller procedure was found to comprise about 70% of the L-threo (2S, 3S) isomer and 30% of the D-erythro (2S, 3R) isomer. This analytical result was obtained by gasliquid chromatography (GLC) of trimethylsilyl derivatives of N-acetylsphingosines which were prepared by enzymatic hydrolysis of synthetic N-acetylsphingosylphos-phorylcholines with Clostridium perfringens phospholipase C. Some other evidence of the different chemical configuration between the erythro and threo isomers of synthetic N-acylated sphingosylphosphorylcholines was also provided by thin layer chromatography (TLC), optical rotatory dispersion (ORD), and fast atom bombardment (FAB) mass spectrometry.View full abstractDownload PDF (819K) -
Takayuki SUGIURA, Naoko SOGA, Hiroko NITTA, Keizo WAKU1983 Volume 94 Issue 5 Pages 1719-1722
Published: November 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit platelets contained significant amounts of alkylacyl compounds (10.4%) in choline phosphoglycerides (CPG) and alkenylacyl compounds (46.3%) in ethanolamine phosphoglycerides (EPG). The fatty chain profiles of alkylacyl and diacyl CPG, and alkenylacyl and diacyl EPG were considerably different from each other. Alkylacyl CPG contained higher amounts of 16:0 (35.0%) and 20:4 (20.9%) than diacyl compounds (4.5% and 12.6%, respectively). On the other hand, alkenylacyl EPG included a large amount of 20:4 (58.2%) in comparison with diacyl analogues (28.8 %).View full abstractDownload PDF (304K)
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