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Volume 92 (1982)
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- Issue 4 Pages 977-
- Issue 3 Pages 607-
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Volume 92, Issue 3
Displaying 1-46 of 46 articles from this issue
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Susumu TSUNASAWA, Kozo NARITA1982 Volume 92 Issue 3 Pages 607-613
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy using a reversed phase HPLC system, we have developed a rapid and quantitative micro-identification method for amino-terminal acetylamino acids in proteins at the sample range of 10-100 nmol.
The process of the identification method consists of the following steps: (1) digestion of a protein by an appropriate protease; (2) separation of an acidic peptide(s) from the digest on a Dowex 50×2 column; (3) further purification of the acidic peptide (s) by reversed phase HPLC using a C18 column; (4) subsequent exhaustive digestion of each acidic peptide to amino acids and an acylamino acid, by pronase and carboxypeptidase Y; (5) isolation of the acylamino acid on a Dowex 50×2 column; (6) identification of the acylamino acid on a C18 column; (7) confirmation of the acyl group by HPLC as an 1-acyl-2-dansylhydrazine derivative and of the amino acid on amino acid analyzer. By applying this new method to such proteins as ovalbumin and cytochrome c, their amino-terminal acetylamino acids can be determined in the range of less than 10 nmol.View full abstractDownload PDF (490K) -
Masahiro IWAKURA, Yukio SHIMURA, Keishiro TSUDA1982 Volume 92 Issue 3 Pages 615-622
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study. The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes. The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites. Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes. Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites. The deletion of these restriction sites using single-strand-specific nuclease SI suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.View full abstractDownload PDF (1167K) -
Hideki TACHIBANA, Shizue UENO-NISHIO, Osamu GOTOH, Akiyoshi WADA1982 Volume 92 Issue 3 Pages 623-635
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHigh-resolution differential melting curves of ØX174 Y1 and Y2 restriction fragment DNAs, for which the base sequences were known, were measured at various sodium ion concentrations ranging from 195 to 2.3mM. The curves were resolved into component peaks, and the change in the melting temperature, the change in the area, and the change in the breadth of each peak with change in salt concentration were examined. The locations of the melting regions corresponding to the peaks in the melting curves were assigned based on theoretical calculations of melting curves and stability maps. It was found that as the salt concentration was decreased from the high to the intermediate range, the breadths of the peaks on the low-temperature side decreased whereas those on the high-temperature side remained almost constant, and also the separation between the peaks along the temperature axis increased. Changes in the positions of peaks relative to one another were interpreted in terms of the difference in the free energy increase between a loop state and an end-coil state as the salt concentration decreased.View full abstractDownload PDF (970K) -
Norio SAKURAGAWA1982 Volume 92 Issue 3 Pages 637-646
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380, 000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis p-nitrophenyl phosphate slowly compared with [14C] sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70, 500 and 39, 800. This enzyme had a molecular weight of 200, 000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6×10-5 M for [14C] sphingomyelin.
Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.View full abstractDownload PDF (1187K) -
Takaaki KOBAYASHI, Gen MATSUMOTO1982 Volume 92 Issue 3 Pages 647-652
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytoplasmic tubulin from squid nerve fully retains C-terminal tyrosine as a result of the almost zero activity of detyrosinating enzyme and low but definite activity of tubulin-tyrosine ligase in its cytoplasm, while the tyrosine content of cytoplasmic tubulin from mammalian brain is about 30 to 60% of that expected for the fully tyrosinated state because of much higher activity of detyrosinating enzyme in mammalian brain. On the other hand, membrane tubulin from both squid optic ganglion and mammalian brain retains only a small amount of C-terminal tyrosine. The significance of these differences is discussed.View full abstractDownload PDF (426K) -
I. Location and Role of Tryptophan ResiduesYoko KITA, Masashi FUKAZAWA, Yasunori NITTA, Takehiko WATANABE1982 Volume 92 Issue 3 Pages 653-659
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Five and four tryptophan residues in Taka-amylase A [EC 3. 2. 1. 1] of A. orvzae (TAA) were modified with dimethyl (2-hydroxy-5-nitrobenzyl) -sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl (2-methoxy-5-nitrobenzyl) -sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose.
Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, K1) of the enzyme modified with K-IWS and K-IIWS were determined. The ko value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3±0.5)×105 M-1, s-1, (2.1±0.3)×103 M-1•s-1 and (1.7±0.2)×102 M-1•s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6±0.6)×105 M-1•s-1 and (6.9±1.1)×104 M-1•s-1, respectively.
On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.View full abstractDownload PDF (530K) -
V. Molecular Properties of Porcine Heart C-Nitrosoreductase and Identity of This Enzyme with NAD (P) H DehydrogenaseShigeo HORIE, Tomino WATANABE, Akishige OHTA1982 Volume 92 Issue 3 Pages 661-671
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNAD (P) H-dependent C-nitrosoreductase of porcine heart cytosol was purified 12, 000-fold in the presence of NADH with an overall yield of 2.2%. The purification procedure included ammonium sulfate fractionation, gel filtration with Sephadex G-100, ion-exchange chromatography on DEAE-Sephadex A-50, hydrophobic chromatography on Octyl-Sepharose CL-4B, and gel filtration with Sephadex G-200. The purity of the preparation was approximately 90% and the molecular weight of the enzyme estimated by gel filtration was about 60, 000. The purified enzyme was composed of two molecular forms, nitrosoreductases 1 and 2, having isoelectric points of 8.45 and 8.6, respectively. A significant amount of zinc was found in the preparation by X-ray fluorescence analysis. The enzyme as it was prepared was colorless, but, after oxidation with p-nitrosophenol followed by gel filtration in the absence of NADH, it showed the absorption spectrum of a flavoprotein. Spectral data indicated the presence of 1 mol of flavin per mol of the enzyme. The molecular turnover number was calculated to be 10, 000 nmol p-nitrosophenol reduced to p-aminophenol per min per nmol enzyme at pH 5.8 and 22°C. The activity was inhibited by p-chloromercuribenzoate by 50% at a concentration of 3×10-5 M. Besides the nitrosoreductase activity, the purified preparation showed NAD (P) Hdependent menadione reductase activity. The activities were both strongly inhibited by dicumarol and markedly activated by serum albumin and by Tween 20. These results indicate the probable identity of this enzyme with soluble NAD (P) H dehydrogenase (quinone) [EC 1. 6. 99. 2].View full abstractDownload PDF (838K) -
Takeo YAMAGUCHI, Sumio KUROKI, Michinori TANAKA, Eiji KIMOTO1982 Volume 92 Issue 3 Pages 673-678
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of temperature and cholesterol on the membrane fluidity of human erythrocytes were studied using 5-nitoxide stearic acid (5NS), 12-nitoxide stearic acid (12NS), and 16-nitroxide stearic acid (16NS).
Human erythrocytes and their lipid vesicles were treated in the range of 5-55°C. In erythrocytes, ESR signals for 12NS and 16NS showed line broadening above 40°C, whereas those for 5NS became sharper with increasing temperature as was the case with the signals of lipid vesicles for each label molecule. Lipid extraction from the heated sample caused no radical reduction. Only in 12NS-labeled erythrocytes did a weakly immobilized component and a strongly immobilized component appear. In the time course at 50°C, the former decreased and the latter remained constant. From the ratio of both components, it was found that the interaction of the label molecules with the binding sites was determined by the physical state of the membrane. Furthermore, the dependence on temperature of the molecular motion of the labels in the cell membrane was irreversible above 40°C.
On addition of cholesterol to the membrane, the outer hyperfine splittings for 12NS and 16NS increased but that for 5NS decreased at C/P>1, perhaps indicating a spread between the head groups of phospholipids by cholesterol.View full abstractDownload PDF (494K) -
Toshio SHIMADA, Teruko SUGO, Hisao KATO, Sadaaki IWANAGA1982 Volume 92 Issue 3 Pages 679-688
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlthough a method for purification of high-molecular-weight (HMW) kininogen from bovine plasma has been reported (Komiya et al. (1974) J. Biochem. 76, 811-822), the preparation of HMW kininogen consisted of a mixture of two molecular species, a single chain kininogen and a two chain kininogen, the latter of which suffered limited proteolysis of the polypeptide chain. We succeeded here in establishing a procedure for preparation of a single chain HMW kininogen. The method consisted of three steps, chromatographies on columns of DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration on a column of Sephadex G-150 in 0.02M Tris-HCI buffer, pH 8.0, containing 1M NaCl. Although HMW kininogen existed as a molecular complex with prekallikrein at a low salt concentration, the complex was dissociated and the components separated from each other by the final step with molecular sieving at a high salt concentration. The kininogen prepared by the present method showed a single band on SDS-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol and on disc polyacrylamide gel electrophoresis at pH 7.2, indicating that it consists of a single chain polypeptide. The single chain kininogen was indistinguishable from the previous preparation, with regard to the amino acid composition and immunological properties. Moreover, no significant difference was observed in their accelerating activities on the kaolin-mediated activation of Factor XII in the presence of prekallikrein. The single chain kininogen was also demonstrated to be free from kallikrein, prekallikrein, Factor XIIa and Factor XII based on the following results; (1) it was not degraded into the two chain protein upon incubation at 37°C for 24 h, (2) it did not hydrolyze carbobenzoxy-phenylalanyl-arginine-4-methylcoumaryl-7-amide, which is a very sensitive substrate for plasma kallikrein, even in the presence of each of kaolin, Factor XII or XIIa and prekallikrein, and (3) it did not correct the prolonged clotting time of Factor XII deficient plasma.View full abstractDownload PDF (1404K) -
Teruko SUGO, Amelia HAMAGUCHI, Toshio SHIMADA, Hisao KATO, Sadaaki IWA ...1982 Volume 92 Issue 3 Pages 689-698
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of kaolin-mediated activation of bovine Factor XII was studied in the presence of prekallikrein and HMW kininogen. The activated enzymes were assayed using fluorogenic peptide substrates, Boc-Glu (OBzl)-Gly-Arg-4-methyl-coumaryl-7-amide (MCA) for Factor XIIa and Z-Phe-Arg-MCA for plasma kallikrein. The rates of activation of the zymogens were separately measured by blocking either of the active enzymes with a specific inhibitor, corn inhibitor for Factor Xlla and Trasylol for plasma kallikrein. The results were as follows:
1. At the early stage of the activation reaction, kallikrein activity was first generated after a short lag phase, and then Factor XIIa activity was generated with a sigmoidal curve. In the presence of corn inhibitor, the activation of prekallikrein was observed, but in the presence of Trasylol, the activation of Factor XII was not observed. In the presence of a high concentration of Ala-Phe-Arg-CH2Cl, which immediately inactivates both of the active enzymes, the cleavage of a single chain prekallikrein into the two chain form by Factor XII was found, as revealed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), using nonlabeled and tritiated prekallikrein.
2. The incubation of Factor XII alone in a quartz cuvette or in the presence of kaolin and HMW kininogen did not result in the activation of Factor XII. The concave upward curve due to autocatalytic activation was not observed even after the addition of Factor XIIa to the Factor XII preparation. Moreover, no limited proteolysis of Factor XII during the incubation with kaolin and HMW kininogen was shown by SDS-PAGE, using 3H-Factor XII.
3. The rates of the activation of prekallikrein by Factor XII and by Factor XIIa were approximately the same at a higher concentration of prekallikrein. However, at a lower concentration of prekallikrein, the activation curve was shown to be sigmoidal and the rate was, much slower, than that with Factor XIIa.
These results indicate that the activation of bovine Factor XII is initiated by the attack of Factor XII on prekallikrein, followed by the reciprocal activation of Factor XII by the plasma kallikrein generated. The autocatalytic activation of bovine Factor XII by Factor XIIa was not demonstrated.View full abstractDownload PDF (1516K) -
Satoshi KATO, Toshikazu NAKAMURA, Akira ICHIHARA1982 Volume 92 Issue 3 Pages 699-708
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relation between changes of insulin receptor and various metabolic responses were studied in adult rat hepatocytes in primary culture. In cells cultured for 3 h without insulin, the number of high affinity sites and the dissociation constant (Kd) of insulin receptor, determined from a Scatchard plot, were 1.05×105 sites/cell and 1.5×10-9 M, respectively. The receptor number increased 2-fold, but the Kd value remained constant during 2-days culture in insulin-free medium (up-regulation). Addition of dexamethasone (Dex), growth hormone, glucagon or triiodothyronine did not change the number of insulin receptors or the Kd value. In contrast, 1-day culture in insulin (1×10-7 M) medium decreased the receptor number by half (downregulation) without change of the Kd value. Short-term responses of glycogenesis, amino acid transport and lipogenesis by insulin increased as the receptor number increased. In these cases, the sensitivity to insulin (Ka: half dose for the maximum response) did not change in cells with different receptor numbers, but the maximum response changed. These results show that hepatocytes, unlike adipocytes, do not have spare receptors of insulin. During down-regulation, the receptor number decreased by only half, but the insulin responses were lost almost completely. The receptor number returned to the normal level after culture in insulin-free medium for 12 h, but recovery of the responses took longer, suggesting that for the insulin response not only change of receptor number, but also other regulatory mechanisms for post-receptor processes, such as desensitization, are involved.View full abstractDownload PDF (779K) -
Tamo FUKAMIZO, Takao TORIKATA, Satoru KUHARA, Katsuya HAYASHI1982 Volume 92 Issue 3 Pages 709-716
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe time-courses of the human lysozyme-catalyzed reaction of chitopentaose were measured by high-performance gel-filtration in comparison with those of hen egg-white lysozyme. Human lysozyme has considerably larger rate constants for the cleavage of glycosidic linkages and transglycosylation than those of hen lysozyme, in agreement with the fact that human lysozyme exhibits a large lytic activity.
It has been reported that binding subsite D in human lysozyme has negative free energy on substrate binding, whereas subsite D in hen lysozyme has unfavorable positive free energy due to the distortion of a sugar residue. The time-courses calculated under the assumption that subsite D in human lysozyme has negative free energy on substrate binding did not fit the experimentally obtained time-courses, even though the combination of values of rate constants in the enzymatic reaction widely varied in the calculation of the time-courses. Thus, it was concluded that subsite D in human lysozyme may not have negative binding free energy, but positive values similar to hen lysozyme.View full abstractDownload PDF (646K) -
Tamo FUKAMIZO, Satoru KUHARA, Katsuya HAYASHI1982 Volume 92 Issue 3 Pages 717-724
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe time-courses of action of Trp 62-modified lysozymes on the initial substrate chitopentaose were measured by means of high-performance gel-filtration. The activities of the modified lysozymes, represented by the rate of disappearance of the initial substrate (overall rate) were lowered to various extents depending on the method of the modification. On the other hand, the time-courses were calculated by changing the values of rate constants, using the binding free energy of each subsite estimated by the optimization technique (Kuhara et al. (1982) J. Biochein.). For NBS- and NPS-lysozymes, the calculated time-courses were not in good agreement with the experimental ones, when the binding free energies estimated by the optimization technique were used for the calculation. Therefore, the binding free energies of the subsites were estimated from the experimental time-courses with the assumption that the values of the rate constants do not change upon modification of Trp 62 in subsite C. As a result, it was found that, though the modification was at subsite C, the binding free energy of subsite A was profoundly lowered, while that of subsite B remained almost unchanged.View full abstractDownload PDF (671K) -
Keikichi TAKAHASHI, Yukio IKEHARA, Keitaro KATO1982 Volume 92 Issue 3 Pages 725-736
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Golgi-rich fraction has been isolated from rat ascites hepatoma AH-130 cells. Unlike the usual procedure for isolating Golgi complexes from liver and other tissues, a hypotonic solution including 2mM CaCl2. was used as the homogenization medium for the ascites hepatoma cells, followed by a combination of differential and discontinuous sucrose gradient centrifugations.
Electron microscopic observation revealed that the isolated fraction consisted of cisternae, vesicles and tubular elements which were similar to those structures described previously for the Golgi fraction isolated from rat liver. Galactosyl- and sialyl-transferases were concentrated about 55- and 75-fold, respectively, in this fraction compared with the homogenate, indicating that these enzymes are useful markers for the Golgi complex of rat ascites hepatoma AH-130 cells, as they are for those of other normal tissues. The preparation was virtually free of cytochrome oxidase, but contained minor amounts of acid phosphatase, alkaline phosphatase and phosphodiesterase I activities.
Electrophoretic analysis on sodium dodecylsulfate-polyacrylamide gels showed that the hepatoma Golgi membranes were resolved into at least 23 protein bands, which were apparently different from the electrophoretic profile of the plasma membrane isolated from the same hepatoma cells.View full abstractDownload PDF (3173K) -
Keikichi TAKAHASHI, Yukio IKEHARA, Masao OGAWA, Keitaro KATO1982 Volume 92 Issue 3 Pages 737-748
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated the Golgi-rich fraction from rat ascites hepatoma AH-130 cells and rat liver, and compared some properties of glycosyltransferases using various acceptors. The specific activity of sialyltransferase in the hepatoma Golgi fractions was reduced to 19-41% depending upon the acceptor used (asialo-orosomucoid, asialo-fetuin or asialo-mucin), as compared to that of the normal liver Golgi fraction. However, no significant difference between the enzymes from the two sources was observed in pH optimum, requirements for the enzyme activity, and Km values for the donor substrate (CMP-sialic acid) and various acceptors used. The specific activity and other kinetic parameters of hepatoma galactosyltransferase were not significantly different from those of the liver enzyme, when assayed with N-acetylglucosamine, asialo-agalacto-fetuin and asialomucin as acceptors.
Glycosyltransferases in the hepatoma and liver Golgi fractions were then assayed with plasma membranes from both sources as exogenous acceptor. Hepatoma sialyltransferase activity was much lower (1/2 to 1/4) than that of the normal liver. Galactosyltransferase activity, however, was found to be slightly higher in the hepatoma Golgi fraction than in the normal liver. Acceptor plasma membranes which were thus glycosylated in vitro by each Golgi enzyme were separated into protein and lipid fractions, and the latter fraction was further analyzed by thin layer chromatography. The results suggest that the hepatoma Golgi had much lower levels of glycoprotein : sialyltransferas and asialo-GM1: sialyltransferase, but had an increased activity of asialo-GM3: sialyltransferase. It is also suggested that the hepatoma Golgi had a high activity for the formation of di- and tri-glycosyl-ceramides, for which the liver Golgi showed negligible activity.View full abstractDownload PDF (2288K) -
Takumi HATAYAMA, Masaaki INABA, Munehiko YUKIOKA1982 Volume 92 Issue 3 Pages 749-755
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have shown that, in rat liver nuclei, the chromatin-bound RNA polymerase II is released as two different forms on digestion with micrococcal nuclease or DNase I (peak 1 and peak 2).
To elucidate the origin of the two forms of the enzyme, we examined their distribution in fractionated chromatins obtained by mild micrococcal nuclease digestion of the nuclei. About half of the total peak 2 activity was recovered in a nuclease-sensitive chromatin fraction which contained DNA enriched in the sequences appearing in the polysomal polyadenylated mRNA. On the other hand, four-fifths of the total peak 1 activity was recovered in a nuclease-resistant chromatin fraction which contained DNA comprising only two thirds of the transcribed sequences. Furthermore, during the nuclease digestion, peak 2 activity was rapidly released from the chromatin, whereas peak 1 activity was gradually released.
These results indicate that the two forms of RNA polymerase II are distributed differently in the cell nuclei.View full abstractDownload PDF (508K) -
Akira WAKIZAKA, Eiji OKUHARA1982 Volume 92 Issue 3 Pages 757-763
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPyrimidine, purine, and mixed sequence oligonucleotides from ultraviolet-irradiated DNA were tested for their inhibitory activities on the interaction of [3H] ultraviolet-irradiated DNA with its antibody raised in rabbit. Thymine dimer containing pyrimidine oligonucleotides from irradiated DNA failed to inhibit the interaction, while mixed sequence oligonucleotides, especially those with 8 or more nucleotides, exhibited potent inhibition. Purine clusters from irradiated DNA and mixed sequence oligomers from unirradiated DNA showed no inhibition. Dimerized thymine, which appears to be a critical part of the antigenic determinant, did not inhibit the interaction by itself. The same observations were made for ultraviolet-irradiated thymidine and thymidylic acid. The results suggest that a structure composed of a mixed pyrimidine and purine sequence with a certain chain length seems to be essential for the antigenicity induced in the irradiated DNA. On this nucleotide chain backbone, photochemically modified bases (mostly thymine dimer) can form an immune-active structure.View full abstractDownload PDF (567K) -
Toshihiko HIRANO, Haruki YAMADA, Toshio MIYAZAKI, Akira TAKATSUKI, Gak ...1982 Volume 92 Issue 3 Pages 765-773
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of tunicamycin (TM) on the biosynthesis of contact site A (cs-A), which is thought to be involved in the cell adhesion of aggregation-competent cells of Dictyostelium discoideum, was investigated. The butanol extract from particles of untreated cells showed a major protein band with a molecular weight of 80, 000 daltons on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). However, this protein band was not detected in the extract from TM-treated cell particles by SDS-PAGE. The monovalent antibodies prepared from the antiserum which was produced against the membrane particles of the aggregation-competent cells (anti P) showed a significant inhibitory activity against EDTA-stable cell adhesion of the aggregation-competent cells. This result indicated that the antibody contained anti cs-A. Upon immunoaffinity chromatography on anti P-immunoglobulin G (IgG)-conjugated protein A/Sepharose, the protein band at 80, 000 daltons corresponding to cs-A was detected in the absorbed fraction of the control extract, but it was not detected and a single protein band at 70, 000 daltons newly appeared in the same fraction from TM-treated cells. When the radioactive antigens in the [3H] leucine-labeled butanol extract from the control or TM-treated cells were further purified by a second affinity chromatography on concanavalin A (con A) Sepharose, the major radioactive peak at 80, 000 daltons was detected in the control, while this peak was not detected in the TM-treated cell extract. However, the minor radioactive peak at 70, 000 daltons which was not detected in the control antigen was detected in the con A-reactive antigens from TM-treated cell extract. This protein at 70, 000 daltons seemed to be incomplete cs-A, lacking asparagine-linked oligosaccharide chains. These results confirmed that the N-glycosylation of an adhesion-mediating glycoprotein, cs-A, was inhibited by the TM treatment of the cells.View full abstractDownload PDF (744K) -
Kenji SUGIYAMA, Toshihiro EMORI, Sumi NAGASE1982 Volume 92 Issue 3 Pages 775-779
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe synthesis and secretion of plasma proteins by isolated hepatocytes from the liver of Nagase analbuminemia rats (NAR) were investigated. NAR hepatocytes did not synthesize any albumin, but showed enhanced synthesis and secretion of several other plasma proteins, including transferrin, hemopexin, ceruloplasmin and fibrinogen. The lag period before the secretion of transferrin in NAR hepatocytes decreased. These findings indicate that the increased concentration of plasma globulins in NAR may be due to their enhanced synthesis and secretion in the liver.View full abstractDownload PDF (347K) -
Shin NAKAMURA, Takashi MORITA, Toshie HARADA-SUZUKI, Sadaaki IWANAGA, ...1982 Volume 92 Issue 3 Pages 781-792
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA clotting enzyme associated with the hemolymph coagulation system of Japanese horseshoe crab (Tachypleus tridentatus) was highly purified from the amebocyte lysate. The method for purification consisted of gel-filtration of the lysate on a pyrogen-free Sepharose CL-6 B column and affinity chromatography of the endotoxin-treated clotting enzyme on a benzamidine-Sepharose 4 B column. Through these procedures, about 3 mg of the purified enzyme was obtained from 70 ml of the lysate and about 390-fold purification was achieved. The purified preparation was found to give a single major band, respectively, on polyacrylamide-gel disc electrophoresis at pH 3.2 in the presence of 6M urea and on sodium dodecyl sulfate (SDS)-gel electrophoresis in the presence and absence of 2-mercaptoethanol. It also gave a single symmetrical peak on QAE-Sephadex A-25 column chromatography.
The molecular weight of the clotting enzyme was estimated to be approximately 42, 000 for the unreduced sample by SDS-gel electrophoresis. For the reduced sample, it was 30, 000, suggesting that the protein consists of plural polypeptide chains bridged by disulfide (s). The Tachypleus clotting enzyme was a glycoprotein, as shown by the positive periodic acid-Schiff reaction for the protein band on SDS-gel and the amino acid analysis.
The purified clotting enzyme transformed Tachypleus coagulogen to coagulin gel and hydrolyzed a chromogenic peptide substrate, Tos-Ile-Glu-Gly-Arg-p-nitroanilide for Factor Xa, liberating p-nitroaniline. The enzyme was sensitive to DFP and benzamidine. It was also inhibited partially by PCMB. Antithrombin III and α2-plasmin inhibitor (α2-antiplasmin) were very effective inhibitors of this enzyme among ten kinds of naturally occurring proteinase inhibitors tested. The clotting enzyme had a restricted specificity towards protein substrates and activated only prothrombin among plasma zymogens including Factor IX, Factor X, fibrinogen, plasminogen and prekallikrein. The cleavage sites of bovine prothrombin for this enzyme were the same Arg-Thr and Arg-Ile linkages as those for Factor Xa, resulting in the formation of a-thrombin.
These results indicate that the horseshoe crab clotting enzyme is a Factor Xa-like serine proteinase rather than a-thrombin. It seems likely that the Tachypleus clotting enzyme is a prototype of mammalian serine proteinases participating in blood coagulation.View full abstractDownload PDF (1420K) -
Toshie HARADA-SUZUKI, Takashi MORITA, Sadaaki IWANAGA, Shin NAKAMURA, ...1982 Volume 92 Issue 3 Pages 793-800
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBasic studies on the chromogenic substrate method for the assay of bacterial endotoxins were performed using Tachypleus hemocyte lysate. From the substrate specificity studies, Tachypleus clotting enzyme was found to prefer tripeptidyl-p-nitroanilide (pNA) having glycine and leucine (valine) in the P2 and P3 positions, and the apparent Km values for these peptidyl-pNA's were in the order of 10-5 M. Coagulogen, a natural substrate of the clotting enzyme, competitively inhibited the amidase-catalyzed hydrolysis of Tos-Ile-Glu-Gly-Arg-pNA with a Kt of 3.8×10-6 M, indicating that the amidase activity is due to catalytic action of the clotting enzyme.
Using the chromogenic substrate, the optimal conditions for the endotoxininduced activation of the latent amidase in Tachypleus hemocyte lysate was examined. As a result, it was shown that the following points should be taken into consideration for the assaying of endotoxins; (1) the endotoxin-induced amidase activity in the lysate occurs optimally at pH 8.0 and at 40°C, (2) the induction requires Mg2+, (3) the endotoxin-induced amidase activity is unstable on prolonged incubation, (4) the sensitivity of the lysate to endotoxin is highly dependent upon its protein concentration, and (5) the latent amidase activity in the lysate is induced by a variety of endotoxin preparations with considerable differences in reactivity. In conclusion, the chromogenic substrate method using Tachypleus hemocyte lysate is valuable for the detection and quantitation of bacterial endotoxins, and under certain conditions the method is fifty-times more sensitive than the Limulus gelation test.View full abstractDownload PDF (612K) -
Yoshito KINOSHITA, Akira MAKITA, Takuji TAKEUCHI1982 Volume 92 Issue 3 Pages 801-808
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGangliosides of B16 mouse melanoma cells were characterized and the effects of 5-bromodeoxyuridine (BrdU) on gangliosides were examined in relation to the alteration of cellular phenotypic expression. In melanotic cells, gangliosides were shown to be composed of both hematosides containing N-acetylneuraminic acid (NeuAc-hematoside) and N-glycolylneuraminic acid (NeuGc-hematoside) as determined by thin-layer chromatography, gas chromatography-mass spectrometry and the use of anti-NeuGc-hematoside antiserum. Gangliosides other than hematosides were not detectable under the conditions of thin-layer chromatography employed.
BrdU treatment of the melanotic cells induced about a two-fold increase in the ratio of NeuGc-hematosides to the total hematosides without any significant change in the cellular ganglioside content. The treatment concomitantly induced a change in cellular morphology, an increase in cell-to-substrate adhesiveness, and the suppression of tyrosinase activity. Amelanotic cells, which showed cellular phenotypes similar to those of BrdU-treated melanotic cells, were demonstrated to have over twice as much NeuGc-hematosides as compared with the melanotic cells.
These results indicate a possible relationship in melanoma cells between the molecular species conversion of sialic acids of hematosides and the alteration of cellular phenotypic expression by BrdU treatment or spontaneous amelanization.View full abstractDownload PDF (2473K) -
Koui TAKAHASHI, Fumio NAKAMURA, Mutsuo OKAMOTO1982 Volume 92 Issue 3 Pages 809-815
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein component that modified the interaction between actin and myosin was released from myofibrils on Ca2+-treatment, accompanying the weakening of Z disks. For its release 10-4 M Ca2+ was required. The presence of the component facilitated the dissociation of thin and thick filaments from myofibrils and delayed superprecipitation of reconstituted actomyosin, independently of Ca2+ concentration. The component is probably a constituent of Z disks. In postmortem muscle, it is possible that the component is released from Z disks with an increased concentration of Ca2+, and that it weakens rigor linkages formed between actin and myosin, giving rise to lengthening of rigor-shortened sarcomeres.View full abstractDownload PDF (524K) -
Toshio MURASE, Nobuhiro YAMADA, Yasuo AKANUMA, Nakaaki OHSAWA1982 Volume 92 Issue 3 Pages 817-821
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTriacylglycerol lipase activities in adrenals and testes of rats were measured and the properties of the enzymes were characterized. The enzymes were extracted from acetone-ether dried powder of the tissues and the crude extracts were applied to a heparin-Sepharose affinity column. The enzymes bound to the column were then eluted at different ionic strengths. Two lipases could be detected in rat adrenals: one was inactivated by the antiserum raised against hepatic triacylglycerol lipase and the other had the properties of lipoprotein lipase. In testes, only lipoprotein lipase could be detected. The results are discussed in connection with the possible functions of these two lipases in organs utilizing lipoprotein-cholesterol for steroid hormone synthesis.View full abstractDownload PDF (333K) -
Akinori SUMI, Kozo HAMAGUCHI1982 Volume 92 Issue 3 Pages 823-833
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe denaturation and renaturation by guanidine hydrochloride of Fc (t) fragment whose interchain disulfide bonds are reduced and alkylated (R. A. Fc (t)) and pFc' fragment of a human myeloma protein (IgGI, κ) were studied using tryptophyl fluorescence. R. A. Fc (t) was found to consist of a slow-unfolding region and a rapid-unfolding region. The denaturation of pFc' was extremely slow. Comparison of the kinetic and equilibrium data of the denaturation of R. A. Fc (t) with those of pFc' indicated that the slow-unfolding region of R. A. Fc (t) corresponds to the CH3 region and the fast-unfolding region the CH2 domain. This was also confirmed by the analysis of the CD and fluorescence spectra for R. A. Fc (t) and pFc' at various concentrations of guanidine hydrochloride. Although the kinetic stability of the CH3 region was much higher than that of the CH2 region, the thermodynamic stabilities of these domains were almost the same; the free energy change of the denaturation in water being about 6 kcal•mol-1. This value is also the same as the value for the CL fragment (Goto, Y. & Hamaguchi, K. (1979) J. Biochem. 86, 1433-1441). It was suggested that the high kinetic stability of the CH3 region in R. A. Fc (t) is due to the strong tendency for the CH3 domains to form a dimer.View full abstractDownload PDF (834K) -
Fujiko SUZUKI, Takashi NAKAJIMA, Kanefusa KATO1982 Volume 92 Issue 3 Pages 835-838
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSS-100 protein, a nervous system-specific protein, was determined in a soluble extract of various rat tissues with a sensitive enzyme immunoassay method, which consisted of a solid-phase with immobilized anti-S-100 antibody and the antibody labeled with β-D-galactosidase from Escherichia coli. The minimum detectable amount of S-100 protein was 3 pg/assay. Central nervous tissues (cerebrum, cerebellum, and brain stem) contained 1.4 to 2.8 μg S-100 protein/mg protein, whereas most of the peripheral tissues contained less than 0.05 μg/ml of the specific protein. However, the level of S-100 protein was high in adipose tissue (0.5-1.1 μg/mg) and in trachea (about 0.5 μg/mg), which involves cartilage. The S-100 protein levels in several tissues were significantly higher in female rats than in males at ages of 5 to 6 weeks.View full abstractDownload PDF (1033K) -
Shintaro KIKUCHI, Takashi KUSAKA1982 Volume 92 Issue 3 Pages 839-844
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBeside de novo fatty acid synthetase, two kinds of fatty acid elongating systems have been found in Mycobacterium smegmatis; they are the malonyl-CoA-dependent, acyl carrier protein (ACP)-requiring system (fatty acid synthetase II, named by K. Bloch's group, 1969) and the acetyl-CoA-dependent ACP-non-requiring one (acetyl-CoA-dependent elongation of fatty acids reported by us, 1977). Now, a third fatty acid elongating system, malonyl-CoA-dependent and ACP-non-requiring, has been isolated from an extract of this microorganisms, separately from each of the previous two elongating systems. Primer specificity and cofactor requirements, especially of pyridine-nucleotide-coenzyme, of the last elongation system also distinguish it from the two previously known systems. All three systems, however, were found in a soluble fraction of M. smegmatis, therefore the regulatory mechanism for these elongation systems should be investigated hereafter.View full abstractDownload PDF (415K) -
Kazuhiro SUZUKI, Sumiko SUZUKI, Tadao TERAO, Toshiaki OSAWA1982 Volume 92 Issue 3 Pages 845-853
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cyclic AMP-binding protein of human erythrocyte membranes was solubilized with 0.3% Triton X-100 in 10mM Tris-HCl (pH 7.4) at 4°C, and purified by DEAE-cellulose column chromatography and affinity chromatography on a cyclic AMP derivative-fixed Sepharose 4 B column.
The purified cyclic AMP-binding protein showed a single band (molecular weight: 49, 000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the band was specifically labeled with a photoaffinity analogue of cyclic AMP, 8-N3-cyclic [2-3H] AMP.
This protein bound 1.6 mol of cyclic AMP per molecule with an association constant of 3.8×109 M-1 and the optimum pH for binding was 7.4. The protein inhibited the activity of a purified protein kinase from human erythrocyte membranes [Suzuki, K., Terao, T., & Osawa, T. (1981) J. Biochem. 89, 1-11], while cyclic AMP restored the enzymic activity. The amino acid composition of this protein was different from those of cytoplasmic cyclic AMP-binding proteins.
These observations indicate that the cyclic AMP-binding protein purified in this work is the regulatory subunit of a membrane bound cyclic AMP-dependent protein kinase.View full abstractDownload PDF (665K) -
Purification and Properties of Magnesium- and Manganese-De Ribonucleases H from Chick Embryo'pendentNamiko KITAHARA, Yasuko SAWAI, Kinji TSUKADA1982 Volume 92 Issue 3 Pages 855-864
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1, 700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40, 000 and of the Mn2+-dependent RNase H about 120, 000, when estimated by gel filtration. Mgt2+-de-pendent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.View full abstractDownload PDF (814K) -
Masaharu KUBO, Yuji MATSUZAWA, Shinji YOKOYAMA, Shoji TAJIMA, Katsunor ...1982 Volume 92 Issue 3 Pages 865-870
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe present data describe the mechanism of the inhibitory effects of human plasma apolipoproteins A-I and A-II on hydrolysis of triglyceride catalyzed by hepatic triglyceride lipase using a substrate of triolein particles stabilized with gum arabic in vitro. The experimental data could well be described by a model in which apolipoproteins bound to the surface of lipid substrate particles inhibited the enzyme reaction. The values of Km obtained were similar with or without inhibitors and the calculated saturation levels of apolipoprotein binding to the lipid were in good agreement with those obtained in independent binding experiments.View full abstractDownload PDF (484K) -
Hirofumi ONISHI, Takeyuki WAKABAYASHI1982 Volume 92 Issue 3 Pages 871-879
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ATP-induced disassembled molecules of chicken gizzard myosin from “thick filaments” have been examined in the electron microscope using the rotary-shadowing technique and have been compared with the myosin molecules disassembled by high concentrations of ammonium acetate without ATP. Both myosin molecules consisted of two globular heads and a long tail. However, two remarkable differences between these myosin molecules were observed:
1. Most of the myosin molecules disassembled by ATP had intramolecular hairpin “loops” in the tails. The length of the hairpin loop was about 510 Å and that of the remaining part of the tail was about 600 Å. On the other hand, no myosin molecules disassembled by ammonium acetate had the intramolecular loop in the tail.
2. The two globular heads of myosin molecules disassembled by ATP tended to bend back towards the tail, but those of the myosin molecules disassembled by ammonium acetate tended to bend forwards.View full abstractDownload PDF (3579K) -
II. Isolation and Structural Studies of Three Glycosphingolipids with Novel Sugar SequencesMutsumi SUGITA, Yumi IWASAKI, Taro HORI1982 Volume 92 Issue 3 Pages 881-887
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree novel glycosphingolipids containing five to seven sugars in their saccharide chains have been isolated from larvae of the green-bottle fly, Lucilia caesar. The structures of these glycolipids were identified by sequential enzymatic degradation with exoglycosidases, methylation analysis, and partial acid hydrolysis to be: Gal-NAcα (1-4) GalNAcβ (1-4) GlcNAcβ (1-3) Manβ (1-4) Glc-Cer, Galα (1-4) GalNAcα (1-4)-GalNAcβ (1-4) GlcNAcβ (1-3) Manβ (1-4) Glc-Cer, and GlcNAcβ (1-4) Galα (1-4) Gal-NAcα (1-4) GalNAcβ (1-4) GlcNAcβ (1-3) Manβ (1-4) Glc-Cer.
It should be noted that one N-acetylgalactosamine in their saccharide chains is α-glycosidically linked to another N-acetylgalactosamine by a 1-4 bond and that this is the first example of the presence of an α (1-4) linked galactosamine unit in nature.
The major fatty acids were normal saturated fatty acids with carbon numbers of 16 to 22. The long-chain bases were dominated by C14 and C16-4-sphingenine homologues.View full abstractDownload PDF (1600K) -
Yasufumi MINAMI, Hiromu MUROFUSHI, Hikoichi SAKAI1982 Volume 92 Issue 3 Pages 889-898
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteraction between porcine brain tubulin and neurofilaments was investigated with reference to network formation in vitro. When a mixture of tubulin and neurofilaments was incubated at 33-34°C, its low-shear viscosity measured with a falling ball viscometer, was far above the sum of viscosities of the separate components and gelation was observed upon increasing the concentrations of both tubulin and neurofilaments above a certain level. Gelation that required Mg2+ and GTP was inhibited by Ca2+, Ca2+-calmodulin or ATP. Antimitotic drugs suppressed gelation at substoichiometric concentrations and cold treatment destroyed the formed gel, which indicated that gelation occurred in conjunction with tubulin polymerization. Assaying by centrifugation revealed that the amount of tubulin co-sedimented with neurofilaments was evidently larger in the presence of neurofilaments than in their absence. Furthermore, electron micrographs showed that a large number of microtubules which were shorter than usual were formed in the presence of neurofilaments. Interestingly, measurements with an Ostwald-type viscometer demonstrated that neurofilaments elevated the viscosity of the tubulin solution in a concentration-dependent fashion. In other words, neurofilaments had the ability to stimulate polymerization of tubulin.
We conclude that polymerization of tubulin with neurofilaments produces threedimensional networks in vitro.View full abstractDownload PDF (2789K) -
Yasuo OGAWA, Nagomi KUREBAYASHI1982 Volume 92 Issue 3 Pages 899-905
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa-releasing action of halothane on fragmented sarcoplasmic reticulum (FSR) from bullfrog and rabbit skeletal muscle was examined to understand the mechanism of Ca release in reference to the etiology of malignant hyperthermia.
Halothane has dual action on FSR: the Ca release and the inhibition of Ca uptake. On addition of halothane to loaded FSR, a rapid Ca release was followed by a sluggish Ca leakage which was probably due to a decreased capacity for Ca uptake. The properties of the rapid Ca release by halothane are similar to those of caffeine. It was inhibited by procaine or high concentrations of Mg2+. It was stimulated by a high concentration of ATP. A low temperature was stimulatory for Ca-releasing action on frog FSR while it was inhibitory on rabbit FSR. The result that caffeine shifted the dose response curve for Ca release by halothane to a steeper relation to a range of much lower concentrations suggests that the action of halothane may not be identical with that of caffeine in spite of many similarities.View full abstractDownload PDF (521K) -
Nagomi KUREBAYASHI, Yasuo OGAWA1982 Volume 92 Issue 3 Pages 907-913
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the effect of halothane on Ca-ATPase activity and its partial reactions of fragmented sarcoplasmic reticulum from bullfrog skeletal muscle (frog FSR) and compared them with the effects of caffeine in order to make it clear whether the effect on ATPase activity is related to the release of Ca or the inhibition of Ca uptake. The effect of halothane on the Ca-ATPase reaction of frog FSR was also compared with that of rabbit FSR.
1. Halothane decreased the apparent affinity for Ca2+ of Ca-ATPase activity of frog FSR. Procaine reversed this effect only slightly. Experiments simultaneously determining the Ca-ATPase activity and the EP level revealed that the drug did not affect the rate of EP hydrolysis, and that its main action must be on the steps leading to EP formation with a reduced apparent affinity for Ca2+. The changes in ATP-ADP exchange rate can be explained largely by these effects. Halothane reduced ATP binding to FSR.
2. With intact rabbit FSR, halothane caused an increase in Ca-ATPase activity in the presence of high concentrations of Ca2+, in addition to reducing the apparent affinity for Ca2+. However, the augmentation by halothane was not observed in the presence of A23187. The effects of halothane on rabbit FSR are essentially similar to those on frog FSR.
3. In contrast to halothane, caffeine increased the Ca-ATPase activity of frog FSR at limited range of Ca2+ concentrations in the presence of 1mM Mg2+. This effect was Mg2+ dependent: in the presence of 4mM Mg2+ no change in ATPase activity was observed. Caffeine exerted little effect if any on the ATP-ADP exchange rate.
4. The results mentioned above and other lines of evidence suggest that the effect of halothane on Ca-ATPase activity or its partial reactions should be related to the inhibition of Ca uptake rather than the Ca releasing action.View full abstractDownload PDF (556K) -
Nagomi KUREBAYASHI, Yasuo OGAWA, Hikaru HARAFUJI1982 Volume 92 Issue 3 Pages 915-920
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn reference to the results of Suko et al. (Biochim. Biophys. Acta (1976) 443, 571-586), we examined if the effect of tetracaine, a local anesthetic, is identical with that of halothane, a general anesthetic, on Ca-ATPase and its partial reactions with fragmented sarcoplasmic reticulum from bullfrog (frog FSR) and rabbit skeletal muscle (rabbit FSR), revealing some effects specific to species as well as common ones.
1. Tetracaine at 1mM decreased Ca uptake activities to about 70%. The extent of the inhibition with frog FSR was similar to that with rabbit FSR. Tetracaine (1mM) caused a sluggish leakage of Ca from loaded FSR, which may be due to a nonspecific increased permeability of the membrane.
2. With frog FSR, tetracaine decreased Ca-ATPase activity to about 70% of the control over the whole range of Ca2+ concentrations without changing the EP level, whereas it increased the ATP-ADP exchange rate. These results are at variance with the results of Suko et al.
3. With rabbit FSR, tetracaine decreased Ca-ATPase activity at Ca2+ concentrations lower than 1μM, while it increased it at higher Ca2+ concentrations. However, Ca-ATPase activity in the presence of 5 μM A23187 was decreased by 1mM tetracaine over the whole range of Ca2+ concentrations. The level of EP decreased over the whole range of Ca2+ concentrations with a lowered apparent affinity to Ca2+. The ATP-ADP exchange rate was decreased at Ca2+ concentrations lower than 1μM, and increased at higher concentrations. These are consistent with the results of Suko et al. 4. Some of the effects of tetracaine can be explained consistently as follows: it shifts the equilibrium between E•ATP•Ca and EP toward E•ATP•Ca, and it decreases the rate of EP decomposition. Tetracaine contrasts with halothane, which largely affects the steps to the formation of E•ATP•Ca by reducing the affinity for Ca2+.View full abstractDownload PDF (469K) -
Kiyokazu OGITA, Emi KUSUNOSE, Kosuke ICHIHARA, Masamichi KUSUNOSE1982 Volume 92 Issue 3 Pages 921-928
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome P-450 was purified from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene. 6-Amino-n-hexyl-Sepharose 4 B column chromatog-raphy of the cholate-solubilized microsomes yielded two cytochrome P-450 fractions, one of which was eluted from the column with 20mM potassium phosphate buffer in the presence of 0.4% cholate and 0.08% Emulgen 913. This fraction was partially purified to a specific content of 4.49 nmol of cytochrome P-450/mg of protein. This P-450 fraction catalyzed myristate ω- and ω-1)-hydroxylation with a turnover rate of 5.0 nmol/nmol of cytochrome P-450 in a reconstituted system containing NADPH-cytochrome c reductase, cytochrome b5 and phosphatidylethanolamine. It had no benzo (a) pyrene hydroxylation activity. The other cytochrome P-450 fraction, which was eluted from the column with 0.1M potassium phosphate buffer in the presence of 0.4% cholate and 0.08% Emulgen 913, was purified to a specific content of 12.0 nmol of cytochrome P-450/mg of protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the final preparation gave a major polypeptide band with a molecular weight of 58, 000. This cytochrome P-450 showed a maximal peak at 448 nm in the carbon monoxide difference spectrum of its reduced form. Its absolute spectrum of the oxidized form had low-spin characteristics. It catalyzed benzo (a) -pyrene hydroxylation with a turnover rate of 3.63 nmol/nmol of cytochrome P-450 in a reconstituted system containing NADPH-cytochrome c reductase and phosphatidylcholine, whereas little myristate hydroxylation activity was detected. The results demonstrate the occurrence of multiple forms of cytochrome P-450 in rabbit kidney cortex microsomes.View full abstractDownload PDF (624K) -
Hisafumi HAKATA, Jun-ichi KAMBAYASHI, Goro KOSAKI1982 Volume 92 Issue 3 Pages 929-935
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphatidylinositol-specific phospholipase C was purified to homogeneity from soluble fraction of bovine platelets by ammonium sulfate fractionation, hydrophobic chromatography, DEAE ion exchange chromatography and gel filtration. The purified enzyme has a narrow pH optimum ranging from 6.5 to 7.5 and the molecular weight of the enzyme was estimated to be 143, 000 by sodium dodecyl sulfate slab gel electrophoresis. The purified enzyme requires Ca2+ strictly for activity, which was markedly enhanced in the presence of arachidonate. No enhancement of the activity was observed in the presence of purified calmodulin. The activity was markedly inhibited in the presence of quinacrine but no inhibition by indomethacin was observed.View full abstractDownload PDF (964K) -
Koichi HIRAGA, Goro KIKUCHI1982 Volume 92 Issue 3 Pages 937-944
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe exchange of glycine carboxyl carbon with CO2, catalyzed by the combination of chicken liver glycine decarboxylase (P-protein) and aminomethyl carrier protein (H-protein) was markedly inhibited by various divalent cations, although extents of inhibition by individual metal ions varied considerably. Cu2+ and Zn2+, at 100 μM, inhibited the reaction almost completely, and the inhibitions by Co2+ and Ni2+ were also significant, while Mg2+ and Mn2+ did not appreciably affect the reaction. The inhibition by Zn2+ was competitive with both bicarbonate and H-protein and noncompetitive with glycine. Of the two reactions involved in the glycine-CO2 exchange, decarboxylation of glycine yielding the H-protein-bound aminomethyl moiety was not significantly affected by 100 μM Zn2+ or Cu2+, but carboxylation of the H-protein-bound aminomethyl moiety to form glycine was strongly inhibited by either Zn2+ or Cu2+. Various degrees of inhibition of the glycine-CO2, exchange by other divalent metal ions could also be accounted for by the inhibition of the carboxylation step of the exchange reaction. The primary site of the action of divalent metal ions is likely to be not P-protein but H-protein, and the binding of metal ions with the H-protein-bound intermediate of glycine decarboxylation was assumed to account for the observed marked inhibition.View full abstractDownload PDF (628K) -
Kazutaka TANIZAWA, Michiharu NAKANO, William B. LAWSON, Yuichi KANAOKA1982 Volume 92 Issue 3 Pages 945-951
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn “inverse substrate” for trypsin, p-amidinophenyl acetylglycinate, has been synthesized and applied to mechanistic studies of tryptic hydrolysis. The compound reacts to form the intermediate acetylglycyl trypsin in a very specific manner, analogous to the reactions of p-amidinophenyl alkanoates with trypsin to give alkanoyl trypsins (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem Soc. 99, 4485). The effects of alkylammonium and alkylguanidinium ions on tryptic hydrolysis of this “inverse substrate” have been investigated, and all alkylamines and alkylguanidines tested behaved as activators, n-Propylamine, n-propylguanidine, and n-butylguanidine, which have been reported to inhibit tryptic hydrolysis of ethyl or p-nitrophenyl acetylglycinate, were activators with p-amidinophenyl acetylglycinate. The activating properties of these amines and guanidines were demonstrated for the first time by making use of the specific characteristics of this inverse substrate. Tryptic mechanisms proposed in the literature are discussed in the light of these observations.View full abstractDownload PDF (560K) -
Tamao ENDO, Keizo INOUE, Shoshichi NOJIMA1982 Volume 92 Issue 3 Pages 953-960
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour glyceroglycolipids containing dipalmityiglycerol were synthesized. The thermotropic behavior and barrier function of aqueous suspensions of these glyceroglycolipids were studied. The gel to liquid crystalline phase transition temperatures of these glycolipids were higher than that of dipalmitoylphosphatidylcholine. The interaction between headgroups in glyceroglycolipids might be stronger than that in dipalmitoylphosphatidylcholine. Diglycosyl dipalmitylglycerols could form liposomes and function as a barrier against water-soluble small molecules (glucose, UmP, and H+) when assayed below their phase transition temperatures, like dipalmitoyl-phosphatidylcholine liposomes. The fundamental properties of glyceroglycolipid membranes so far examined were rather similar to those of phospholipid membranes, with the exception that the permeability rate of water through glycolipid membranes was higher than that through phospholipid membranes. This property might cause the apparent sensitivity difference between liposomes prepared from glycolipids and those from phospholipids to amphotericin B.View full abstractDownload PDF (651K) -
Joseph O. FOLAYAN1982 Volume 92 Issue 3 Pages 961-966
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly (8-oxyinosinic acid) (poly O8I), was synthesized by polymerizing 8-oxyinosine diphosphate using the enzyme polynucleotide phosphorylase (PNPase). The polymer formed a 1:1 hybrid with polycytidylic acid (poly C). The hybrid was found to induce the production of interferon in the brain of white albino mice and protected mice against Wesselsbron virus (H 10964).View full abstractDownload PDF (374K) -
Kenzo OHTSUKI, Samuel BARON1982 Volume 92 Issue 3 Pages 967-970
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTreatment of mouse L cells with mouse IFNγ induced a cytoplasmic Ca-dependent protein kinase, which highly phosphorylated cellular enzymes such as phosphodiesterase and RNase in vitro. The kinase partially purified from IFN, -treated cells (100 units/ml, 12 h at 37°C) was different from IFN-induced dsRNA-dependent protein kinase since it was dsRNA independent. The kinase may have played an important role in mediating IFN-induced biological effects, since cellular enzymes were found to alter enzyme activity after phosphorylation by the kinase in vitro.View full abstractDownload PDF (348K) -
Hiroko KATAOKA, Mutsuo SEKIGUCHI1982 Volume 92 Issue 3 Pages 971-973
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme activity to incorporate labeled dATP or dGTP preferentially into depurinated DNA, found in an extract of Escherichia coli, does not seem to represent “purine insertase, ” but may be ascribed to a combined action of DNA polymerase I and AP endonuclease (s) on the basis of the following findings. (1) The activity was found in polA+ but not in polA- cells. (2) The base moiety and the a-phosphate group of the nucleotide were equally incorporated into the DNA.View full abstractDownload PDF (218K) -
1982 Volume 92 Issue 3 Pages 975a
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (34K) -
1982 Volume 92 Issue 3 Pages 975b
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (34K)
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