All issues
Volume 96, Issue 1
Displaying 1-35 of 35 articles from this issue
- |<
- <
- 1
- >
- >|
-
Yoshimasa ITO, Yoshihito WATANABE, Kazuyuki HIRANO, Mamoru SUGIURA, Sh ...1984 Volume 96 Issue 1 Pages 1-8
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new method for the measurement of urinary dipeptidase activity is described. The action of dipeptidase on L-Ala-L-Ala results in production of an L-alanine, and this amino acid is simultaneously determined by an L-alanine dehydrogenase-diaphorase system. As urinary substances do not affect this reaction, the measurement can be accomplished without prior dialysis. The mean value±S. D. for normals was found to be 12.0±4.4 IU/g of creatinine. Elevated values were found in chronic nephritis (55.9±35.0 IU/g of creatinine, P<0.001 vs. normal), acute nephritis (46.6±29.9 IU/g of creatinine, P<0.001), and nephrotic syndrome (43.3±36.5 IU/g of creatinine, P<0.001). The dipeptidase activity thus measured showed a significant correlation with dipeptidase activity against L-Leu-L-Leu as substrate. On disc polyacrylamide gel electrophoresis, the urinary dipeptidase of a patient with chronic nephritis appeared as one band with similar mobility to human kidney dipeptidase F. Urinary dipeptidase in a patient with chronic nephritis was identical to human kidney dipeptidase on double immunodiffusion analysis.View full abstractDownload PDF (1017K) -
Masato HIRATA, Toshihiko HASHIMOTO, Takafumi HAMACHI, Toshitaka KOGA1984 Volume 96 Issue 1 Pages 9-16
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe changes in the intracellular free Ca2+ in guinea pig peritoneal macrophages induced by N-formyl chemotactic peptides were examined using a fluorescent Ca2+-indicator, quin 2. The ATP contents of quin 2-loaded macrophages were also examined. The intracellular free Ca2+ was immediately raised about 4-fold by the addition of chemotactic peptides both in the presence and absence of extracellular Ca2+, and returned to the basal level within 6min. A mitochondrial uncoupler had no effect on basal free Ca2+ concentration and the increase in intracellular free Ca2+ induced by chemotactic peptides. A 23187 increased the intracellular free Ca2+ concentration and minimized the increase by chemotactic peptides. Chlorpromazine also gradually increased the basal level, in agreement with our previous report that this drug induced Ca2+ release from the store sites. The results indicate that the increase in the intracellular free Ca2+ induced by chemotactic peptides is due to Ca2+ release from the membraneous store site (s), other than mitochondria. Extracellular Ca2+ was raised by the addition of a chemotactic peptide, when assayed in Ca2+-free saline using quin 2. The second addition of the chemotactic peptide, after the intracellular free Ca2+ concentration had returned to the basal level, was ineffective. Recovery of the free Ca2+ change induced by chemotactic peptide was observed only when the macrophages were freshly incubated in Ca2+-containing saline for more than 20min at 37°C. These results suggest that the Ca2+ released from the store site (s) may be effluxed through the plasma membrane.
Quin 2 loaded in macrophages may interfere with mitochondrial ATP synthesis.View full abstractDownload PDF (596K) -
Sadako YAMAGATA, Tatsuya YAMAGATA1984 Volume 96 Issue 1 Pages 17-26
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe FBJ osteosarcoma (a virus-induced osteosarcoma named after its discoverers, Finkel, Biskis, and Jinkins) contains an extensive extracellular matrix. Collagens were extracted by digestion with pepsin in dilute acetic acid from tumors grown in lathyritic mice and fractionated by differential salt precipitation, yielding five fractions. Fraction 1 (precipitated at acidic 0.7M and neutral 2.0M NaCl) gave rise mainly to α1 (III) chain on phosphocellulose column chromatography. The α1 (III) chain was identified by its typical behavior on interrupted electrophoresis and analysis of the CNBr-cleaved peptides. The α1 (III) chain of the FBJ tumor had a high content of hydroxylysine and neutral saccharide. Fraction 2 (precipitated at acidic 0.7M and neutral 4.5M NaCl) yielded α1 (I) and α2 (I) chains on the phosphocellulose column from which α1 (I) was eluted as a broad peak, conceivably reflecting a high content of hydroxylysine and neutral saccharide. Fraction 4 (precipitated at acidic 1.2M and neutral 4.5M NaCl) yielded type V collagen, which also featured an exceptionally high content of neutral saccharide (Yamagata, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 1208-1214). The proportions of type I, type I trimer, type III, and type V collagens extracted by pepsin digestion from FBJ tumor were calculated to be 33, 29, 26, and 12%, respectively.
The FBJ tumor is free from invasion by blood vessels, shows no deposition of calcium, and thus has the appearance of cartilage. But type II collagen, a specific gene product of cartilage, could not be identified in any of the fractions analyzed. Contrary to its appearance, collagen type analyses indicate that FBJ osteosarcoma is literally induced from osteogenic cells.View full abstractDownload PDF (2965K) -
VIII. Thiols of MyosinTakamitsu SEKINE, Seitaro TAKAHASHI, Setsuko HIKITA, Noriko SUTOH, Kaz ...1984 Volume 96 Issue 1 Pages 27-33
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin has 2 mol of the most reactive thiol, named SH1. 1, 2, 4-Trinitrobenzene (TNB), a novel dinitrophenyl (DNP) ating reagent [Takahashi et al. (1983) Chem. Lett. 1445-1448], was found to react only with SH1 without any other amino acid residues in myosin under the conditions used. Its reaction with myosin SH1 was about 30 times faster than that with N-acetylcysteine (NAC). The reaction rate of TNB with SH1 was about twice compared with that of NEM, the most reactive and selective reagent for SH1 so far found, although its rate with NAC was only one sixtieth that of NEM. As to the λmax, of the absorption spectrum of SH1-DNP-myosin, a large red shift of as much as 20 nm was observed compared with low molecular S-DNP derivatives. This red shift disappeared in 8M urea.
This outstanding feature of SH1 modification with TNB was discussed in terms of affinity labeling by interaction with an aromatic amino acid near SH1.View full abstractDownload PDF (518K) -
Jung-Yaw LIN, Tze-Bin CHOU1984 Volume 96 Issue 1 Pages 35-40
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lectin was purified from edible mushroom, Volvariella volvacea by extraction with 5% cold acetic acid in the presence of 0.1% 2-mercaptoethanol, followed by ammonium sulfate fractionation, and DEAE-C-52 and CM-C-52 column chromatographies. The molecular weight was measured to be 26, 000, and the lectin consisted of two non-identical subunits as demonstrated by gel filtration and polyacrylamide gel electrophoresis.
The lectin does not contain half-cystine, methionine, or histidine. The LD50 of the lectin is 17.5mg per kg body weight of mice. The lectin has a moderate inhibitory effect on the growth of tumor cells.View full abstractDownload PDF (875K) -
Demonstration of a Proliferation Regulatory Factor (PRF) in Normal Human Urine, and Its PurificationSeiichiro MORI, Toshio ONISHI, Mutumi MURAMATU1984 Volume 96 Issue 1 Pages 41-50
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA material (s) affecting the proliferation of eukaryotic cells, such as C3H2K cells, and prokaryotic cells, such as E. coli, was found in the urine of normal healthy men and was concentrated in a certain fraction, “lysin-Sepharose eluate, ” of the urine. The material was named proliferation regulatory factor, PRF. With increase in the concentration of PRF, the proliferation rate of normal eukaryotes and prokaryotes increased to maxima of no more than twice those of untreated cells. On further increase in the concentration of PRF the growth rates of these cells decreased to the rates of untreated cells. PRF enhanced the rates of synthesis of DNA and RNA in C3H2K cells about 2-fold, but strongly suppressed the rates of growth, DNA synthesis, and RNA synthesis of cancerous cells, such as HeLa and MH 134/M cells.
PRF was purified from the “lysine-Sepharose eluate” by DEAE-cellulose column chromatography and gel filtration on a Sephadex G-100 column. PRF consisted of three molecules with molecular weights of 41, 000, 7, 800, and 5, 100, which were named PRF-I, II, and III, respectively. The lysine-Sepharose eluate and purified PRF's had similar effects on the proliferation of eukaryotes and prokaryotes.View full abstractDownload PDF (714K) -
Masayuki HIRADO, Michio NIINOBE, Setsuro FUJII1984 Volume 96 Issue 1 Pages 51-58
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLarge amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90, 000, and the other a molecular weight of 10, 500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. The higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.View full abstractDownload PDF (1205K) -
Takashi TAKAGI, Kazuhiko KONISHI1984 Volume 96 Issue 1 Pages 59-67
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the β chain of shrimp SCP has been determined. It is composed of 192 amino acid residues and is acetylated at the N-terminus. The molecular weight was determined to be 21, 960. The sequence difference with the αB chain of the same shrimp, of which the sequence was determined previously (J. Biochem. (1984) 95, 1603-1615), is 19% (37 non-identical residues out of 192 residues). The shrimp SCPs have three EF-hand type Ca2+ binding sites, however, from comparison with the amino acid sequences of SCPs of scallop (Takagi et al., Biochim. Biophys. Acta in press) and of sandworm (Kobayashi et al., manuscript in preparation), it is reasonable to think that SCP originally had four Ca2+ binding sites, and in the case of shrimp SCPs, one of them (site IV) may have lost the affinity to Ca2+ on amino acid replacements during evolution.View full abstractDownload PDF (632K) -
Kazuo SAKAGUCHI, Ken'ichiro MITSUI, Noboru NAKAI, Kyoichi KOBASHI1984 Volume 96 Issue 1 Pages 69-72
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCysteine residues in the active center of jack bean urease [EC 3. 5. 1. 5] were modified with 14C-labeled diazonium-1 H-tetrazole (DHT). The labeled enzyme was carboxymethylated with iodoacetic acid, and then hydrolyzed with trypsin. The tryptic digest was subjected to gel filtration on Sephadex G-50, yielding two radioactive fractions. The [14C] DHT-labeled peptide having a lower molecular weight, which was determined to be approximately 1, 000 by the method of gel filtration, was further purified to homogeneity by ion-exchange chromatography on DEAE-Sephadex A-25. [14C] DHT-labeled cysteine was identified as cysteic acid after performic acid oxidation, and the amino acid sequence of the low-molecular-weight [14C] DHT-labeled peptide was determined to be Phe-Glu-Pro-Gly-Asp-Cys-Asn-Ser-Thr-Phe-Lys.View full abstractDownload PDF (778K) -
Kazuo SAKAGUCHI, Ken'ichiro MITSUI, Noboru NAKAI, Kyoichi KOBASHI1984 Volume 96 Issue 1 Pages 73-79
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCysteine residues in the active center of jack bean urease [EC 3. 5. 1. 5] were modified with 14C-labeled diazonium-1 H-tetrazole (DHT). The labeled enzyme was carboxymethylated with iodoacetic acid, and then hydrolyzed with trypsin. The tryptic digest was subjected to gel filtration on Sephadex G-50, yielding two radioactive fractions. The [14C] DHT-labeled peptide having a lower molecular weight, which was determined to be approximately 1, 000 by the method of gel filtration, was further purified to homogeneity by ion-exchange chromatography on DEAE-Sephadex A-25. [14C] DHT-labeled cysteine was identified as cysteic acid after performic acid oxidation, and the amino acid sequence of the low-molecular-weight [14C] DHT-labeled peptide was determined to be Phe-Glu-Pro-Gly-Asp-Cys-Asn-Ser-Thr-Phe-Lys.View full abstractDownload PDF (521K) -
Wataru SAKAMOTO, Katsumi YOSHIKAWA, Soichiro UEHARA, Osamu NISHIKAZE, ...1984 Volume 96 Issue 1 Pages 81-88
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLow molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6 B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72, 000 by SDSPAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin (T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120 T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 μg/ml by a quantitative single radial immunodiffusion test.View full abstractDownload PDF (2139K) -
Masachika IRIE, Fumiko MIKAMI, Kumiko MONMA, Kazuko OHGI, Hideaki WATA ...1984 Volume 96 Issue 1 Pages 89-96
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU>p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25°C.
2. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n≥2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1) +(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length.
3. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.View full abstractDownload PDF (641K) -
Hideo UTSUMI, Tamiko SUZUKI, Keizo INOUE, Shoshichi NOJIMA1984 Volume 96 Issue 1 Pages 97-105
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relation between the immune reactions of phosphatidylcholine liposomes containing galactosyl ceramide and the physical properties of the glycolipid in membranes was studied. The immune-agglutination of dipalmitoyl phosphatidylcholine liposomes was affected both by reaction temperature and by cholesterol content. Fatty acyl chain length of phosphatidylcholine also influenced the immune-agglutination.
The electron spin resonance and calorimetric studies indicated that the fatty acyl chain length of phosphatidylcholine and cholesterol content, as well as temperature, affect the physical properties of galactosyl ceramide in liposomal membranes. In the absence of cholesterol, most galactosyl ceramide molecules were clustered on the phosphatidylcholine liposomes below the chain-melting transition temperature of the phospholipid, whereas they were randomly distributed in the membrane above the transition temperature. Upon addition of cholesterol to the membranes below the chain-melting transition temperature, the number of glycolipid molecules in the cluster phase decreased. Cholesterol increased the ordering of galactosyl ceramide molecules in the phase of random distribution on membranes above the transition temperature. The change in topographical distribution of galactosyl ceramide in membranes was parallel with that of immune-reactivity.View full abstractDownload PDF (673K) -
Hidekazu ARATANI, Shuji KAWATA, Shigeru TSURUYAMA, Norio YOSHIDA, Sato ...1984 Volume 96 Issue 1 Pages 107-115
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn aminopeptidase was purified about 4, 000-fold from the clarified homogenate of bovine leukocytes by a series of column chromatographies on DEAE-cellulose, hydroxyapatite, Sephadex G-150, and DEAE-Toyopearl. The purified enzyme had a specific activity of 3.8 μmol•min-1•mg-1 with arginine β-naphthylamide (Arg-2-NNap) as substrate, and a minute amount of contaminating protein was found to be present by gel electrophoresis. The molecular weight of the enzyme was estimated to be 94, 000 by gel filtration on Sephadex G-150. The enzyme had a broad substrate specificity and a pH optimum between 6.5 and 7.0 for the hydrolysis of α-aminoacyl β-naphthylamides. It hydrolyzed β-naphthylamides of basic, aliphatic, and aromatic amino acids, and also catalyzed the liberation of amino-terminal phenylalanine from phenylalanyl peptides. The enzyme was inhibited by bestatin, puromycin, 1, 10-phenanthroline, sulfhydryl reagents, and a variety of heavy metal ions. Only the cobaltous ion stimulated the enzyme and the values of both Km and Vmax for Arg-2-NNap increased. In gross properties the present enzyme resembles porcine liver aminopeptidase reported previously (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101) very closely.View full abstractDownload PDF (682K) -
Toshiyuki SAKAKI, Ayuko SOGA, Yoshiyasu YABUSAKI, Hideo OHKAWA1984 Volume 96 Issue 1 Pages 117-126
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree forms of cytochrome P-450, designated as P-450MC-I, P-450MC-II, and P-450MC-III, were isolated from liver microsomes of rats treated with 3-methyl-cholanthrene (MC) by using a high performance liquid chromatography (HPLC) technique. The major MC-inducible forms, P-450MC-I and P-450MC-II showed a single protein band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56, 000 daltons. The oxidized absolute spectra of both cytochromes P-450 were of low spin type, having a Soret absorption peak at 417 nm. The CO-reduced difference spectra of these two cytochromes P-450 showed a peak at 447 nm. In a reconstituted system, both cytochromes P-450 exhibited similar high levels of catalytic activity for benzo (a) pyrene hydroxylation and 7-ethoxycou-marin O-deethylation. Anti-P-450MC-I IG and anti-P-450MC-II IG, which were produced against the corresponding cytochromes P-450, each formed a single continuous precipitin line with both P-450MC-I and P-450MC-II in Ouchterlony double diffusion tests. Amino acid sequence analysis revealed that the sequence of the NH2-terminal 18 amino acids of both enzymes was the same. Therefore, the major MC-inducible forms, P-450MC-I and P-450MC-II, were highly homologous, being indistinguishable from each other in terms of apparent molecular weight, spectral properties, substrate specificity and the NH2-terminal 18 amino acid residues, but clearly separable by HPLC. The characteristics of both P-450 forms appear to correspond to those of the previously reported P-450c (1).
On the other hand, a minor form, P-450MC-III was different from P-450MC-I and P-450MC-II in chromatographic properties, apparent molecular weight, substrate specificity and immunochemical properties, and did not correspond to any P-450 species previously purified from MC-treated rat liver microsomes.View full abstractDownload PDF (1708K) -
Toshikazu NAKAMURA, Akito TOMOMURA, Satoshi KATO, Chiseko NODA, Akira ...1984 Volume 96 Issue 1 Pages 127-136
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe stimulations of cyclic AMP formation and adenylate cyclase activity by glucagon and isoproterenol were both found to be highest in neonatal rat hepatocytes and to decrease during development. Adult hepatocytes still showed a considerable response to glucagon, but a negligible response to isoproterenol. The decrease in cyclic AMP formation during development can be explained in the case of the response to β-adrenergic agonist as due to decrease of its receptor number, judging from binding of [125I] iodocyanopindolol to purified plasma membranes. But in the case of the glucagon response, the decrease in the response may be due to change of post-receptor components of the adenylate cyclase system, because the receptor number tended to increase during development, as shown by binding of [125I] iodoglucagon. Similarly, α1-adrenergic receptors increased in number during development, but their IC50 value did not change, as measured by binding of [3H] prazosin to plasma membranes. Previous studies on primary cultures of adult rat hepatocytes showed that the β-adrenergic response and its receptor number increased markedly during short-term culture (Nakamura, T., Tomomura, A., Noda, C., Shimoji, M., & Ichihara, A. (1983) J. Biol. Chem. 258, 9283-9289). However, in this work the amount of α1-adrenergic receptor of adult rat hepatocytes was found to decrease by one third during 1-2 days culture. Therefore, changes of α1- and β-adrenergic receptors during development of rat liver and during primary culture of adult rat hepatocytes were reciprocal, although the directions of change in the two conditions were opposite. The additions of various hormones to primary cultures of adult rat hepatocytes did not affect the reciprocal changes of adrenergic receptors, suggesting that these hormones did not regulate the changes of the receptors.View full abstractDownload PDF (808K) -
Tetsuyuki KOBAYASHI, Hiroshi HOMMA, Yumiko NATORI, Ichiro KUDO, Keizo ...1984 Volume 96 Issue 1 Pages 137-145
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo kinds of E. coli K-12 mutants for lysophospholipase L2 (located in the inner membrane) were isolated, using an improved version of the colony autoradiographic method developed by Raetz; these were, 1) strains carrying an elevated level of the enzyme and 2) strains defective or temperature-sensitive in the enzyme. Characterization of the crude lysates of these mutants revealed that the differences of lysophospholipase L2 activity are not due to the presence or absence of regulatory factors. Evidence was obtained, by using these mutants, that this lysophospholipase L2 transfers the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol, forming acyl phosphatidylglycerol.View full abstractDownload PDF (1343K) -
Toshiaki HIRATSUKA1984 Volume 96 Issue 1 Pages 147-154
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibose-modified fluorescent nucleotide analogs, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives of AT (D) P, dAT (D) P, CT (D) P, UT (D) P, IT (D) P, and GT (D) P, were synthesized for use as substrates and affinity labels for the myosin ATPase [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. None of the fluorescent nucleoside triphosphate (NTP) analogs was significantly different from the corresponding natural NTP in its ability to support superprecipitation of actomyosin. When fluorescent and natural NTPs were used as substrates for the myosin subfragment-1 (S-1) ATPase in the presence of 1mM vanadate ion (V1), a slight initial inhibition of the S-1 NTPase was followed by progressive inhibition to more than 60% over a period of 1h. The apparent second-order rate constants were 0.14-0.44M-1•s-1, suggesting the formation of the inactive fluorescent NDP-labeled S-1. After incubation of S-1 with the nucleoside diphosphate (NDP) analog in the presence of V1, the resultant fluorescent NDP-labeled S-1 was isolated free of unbound V1 and the analog by gel filtration. The isolated complexes had stoichiometries of 0.6-1.1 NDP analog per S-1 active site. Native polyacrylamide gel electrophoresis revealed conveniently that the NDP analog is associated with S-1 as indicated by two intense fluorescent bands corresponding to S-1 isozymes. On dissociating gels, the analog was released from S-1, suggesting that the labeled S-1 is held together by strong secondary forces rather than covalent bonds. Neither nucleoside monophosphate analogs nor P1 could substitute for NDP analogs or V1, respectively, for the labeling. Labeling was abolished by excess ATP and ADP but not by AMP. The results indicate that these nucleotide analogs and V1 function together as fluorescent affinity labels for the myosin active site. It is further concluded that the pyrophosphate bond of NDP is essential for the formation of the stable myosin-NDP-V1 complex but its 2'- and 3'-hydroxyl groups are not.View full abstractDownload PDF (1098K) -
Toshiaki HIRATSUKA1984 Volume 96 Issue 1 Pages 155-162
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active site of the myosin subfragment-1 ATPase was affinity-labeled with ribosemodified fluorescent analogs of ADP, dADP, CDP, UDP, IDP, and GDP in combination with vanadate, forming a stable myosin-nucleoside diphosphate-vanadate complex that is analogous to the normal myosin-ADP-P1 intermediate [Hiratsuka, T. (1984) J. Biochem. 96, 147-154]. Labeled enzyme was isolated free of unbound analog and vanadate, and fluorescent properties of the fluorophore at the active site were examined. Fluorescence emission and acrylamide quenching studies revealed that the hydrophobicity of environment around the fluorophore and the degree of its burial in the protein vary with the base structure of NDP. It was found that the fluorophore of ADP analog is most buried into the protein, while that of the GDP analog is least buried. The results suggest that the deep burial of ATP into the myosin active site is essential for muscle contraction.View full abstractDownload PDF (583K) -
Mamoru ISEMURA, Yu YAMAGUCHI, Hiroshi MUNAKATA, Junichiro AIKAWA, Miki ...1984 Volume 96 Issue 1 Pages 163-169
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFibronectin was isolated from human placenta tissues and compared with human plasma fibronectin. Placenta and plasma fibronectins had similar amino acid compositions, immunological properties, and cell attachment-promoting activities, but differed in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be accounted for at least partly by the difference in carbohydrate composition. Unlike plasma fibronectin, placenta fibronectin failed to form a precipitin line with concanavalin A in a double diffusion system. The non- or low-reactivity of placenta fibronectin with this lectin was also demonstrated by affinity chromatography with concanavalin A-agarose, in which more than 90% of the radiolabeled glycopeptides derived from placenta fibronectin was not retained on the gel. The two fibronectins also differed in the reactivity with Lens culinaris agglutinin of their glycopeptide fractions. These data indicate that placenta and plasma fibronectins are different in their carbohydrate structures and, therefore, suggest the presence of a tissue- or cell-specific mechanism for processing the carbohydrates of this glycoprotein.View full abstractDownload PDF (1625K) -
Kazuhiko WAKABAYASHI, Tadashi MABUCHI1984 Volume 96 Issue 1 Pages 171-177
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA fragment of yeast mitochondrial DNA, Alu B, has two subfragments, Alu B 1 and Alu B 2. They were each cloned and sequenced. The autonomously replicating function of the curtailed Alu B 1 (342 bp) was defined within 186 bp. A GC-rich sequence identical to the oris sequence in the curtailed Alu B 1 was unnecessary for its autonomously replicating function. The 186 bp sequence had an ATATAAAT sequence and the stem and loop structures.
The base sequence of Alu B 2 also contained the same octanucleotides, the stem and loop structures, one oris sequence and one unique GC cluster. Yeast transformants with cloned Alu B 2 grew slowly. The cloned Alu B 2 was enlarged in the yeast host concomitantly with compensation of the slow growth of the transformants.View full abstractDownload PDF (888K) -
Takahisa TAGUCHI, Michiki KASAI1984 Volume 96 Issue 1 Pages 179-188
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lipophilic anion tetraphenylboron (TPB-) but not the lipophilic cation tetraphenylphosphonium (TPP+) increased the choline permeability of isolated sarcoplasmic reticulum (SR). Choline permeability was mainly measured by the stopped flow method by following the change in scattered light intensity. TPB- and TPP+ did not affect the choline permeabilities of liposomes, liver microsomes, or denatured SR vesicles. These phenomena are similar to the Ca2+ release phenomena activated by TPB- reported by Shoshan, MacLennan, and Wood (J. Biol. Chem. 258, 2837 (1983)). These results strongly suggest that TPB- activates a pre-existing channel of SR membrane and choline permeates through the same channel as that for the Ca2+ release. This channel is different from that for the Ca2+-induced Ca2+ release. The former is present in all of the vesicles formed by fragmented SR, while the latter is rich in the heavy fraction of fragmented SR and poor in the light fraction. The channel specificities for permeable ions are different from each other. For example, the latter passes Tris+ but the former does not. The physiological role of this channel is not clear at present.View full abstractDownload PDF (740K) -
Taro AKIBA, Koichi HIRAGA, Syozo TUBOI1984 Volume 96 Issue 1 Pages 189-195
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals.
The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109 A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria.
The mitochondrial fumarase was predominantly located in the matrix. About 10%. of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.View full abstractDownload PDF (556K) -
Retsu MIURA, Hiromasa TOJO, Shigeru FUJII, Toshio YAMANO, Yoshihiro MI ...1984 Volume 96 Issue 1 Pages 197-206
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction between riboflavin and riboflavin binding protein (RBP) was studied by 13C-NMR spectroscopy. The 13C-NMR spectra of riboflavin selectively enriched at the 2-, 4-, 4 a-, and lO a-positions and of (3-[13C] methyl) riboflavin were measured both in the free and RBP-bound forms. The 13C signals of 13C-enriched riboflavin or 3-methylriboflavin bound to RBP are broader than those of the free form, reflecting the restriction of flavin mobility. The 2-, 4-, and 10 a-13C signals of riboflavin show no pH-dependent shift in the neutral to acidic pH region either in the bound or free form but the 4 a-13C signal of bound riboflavin shifts to lower field in the acidic pH region while that of the free form remains unshifted. The 2-, 4-, 4 a-, and 10 a-13C signals of free riboflavin exhibited pH-dependent change in the alkaline pH region with a pK value of about 10, in association with the N (3)-H deprotonation. The pH titration profile of the 2-, 4-, and 4 a-13C signals of bound riboflavin indicates that the pK of N (3)-H is shifted substantially to the alkaline side when riboflavin is bound to RBP. The 3-methyl-13C signal of 3-methylriboflavin shows no pH-dependent shift whether the compound is free or bound to RBP. The binding of riboflavin and 3-methylriboflavin was also studied spectrofluorometrically. The analysis of the pH dependence of the association constant revealed that one ionizable group in RBP with pK of about 5 and N (3)-H of riboflavin play important roles in the binding. We conclude that RBP preferentially binds the neutral, i.e., N (3)-protonated, form of riboflavin and that the neutral form in turn is stabilized by the hydrophobic environment of RBP surrounding the N (3) region of the bound riboflavin molecule.View full abstractDownload PDF (721K) -
Mitsuru NAKAMURA, Shinsei GASA, Akira MAKITA1984 Volume 96 Issue 1 Pages 207-213
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArylsulfatase A was purified from human lung to apparent homogeneity as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enzyme from normal lung as well as that from lung adenocarcinoma showed considerable microheterogeneity when examined by isoelectric focussing, with an isoelectric point (pI) ranging from 5.1 to 4.6. The tumor enzyme was more heterogeneous and contained more acidic components than the normal lung enzyme.
The cause of the charge heterogeneity was examined by treatment with exogenous hydrolases. Upon treatment with sialidase, phosphatase or endo-β-N-acetylglucosaminidase H (endoglycosidase H), the acidic enzyme forms shifted to an alkaline region on isoelectric focussing gels. Combined treatment of the arylsulfatase A with endoglycosidase H and sialidase resulted in complete loss of the most acidic components to give the less acidic components with pI 5.1, 5.0, and 4.9. These results strongly suggest that the charge heterogeneity of arylsulfatase A is due not only to sialylation but also to phosphorylation at the carbohydrate moiety of the enzyme, and the extent of substitution by acidic groups is markedly increased in the tumor enzyme.View full abstractDownload PDF (3134K) -
Junko SOKAWA, Noriaki SHIMIZU, Yoshihiro SOKAWA1984 Volume 96 Issue 1 Pages 215-222
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS(2'-5') Oligoadenylate synthetase (2-5 A synthetase) was found in avian erythrocyte lysates from chicken, goose, and pigeon, with high levels being observed in chicken erythrocytes. No activities, however, were detected in erythrocytes from human, sheep, mouse, turtle, frog, trout, or lamprey. In chicken erythrocyte lysate, about 70% of ATP was converted to 2-5 A molecules during a 20-h incubation, in which the tri- and tetra-adenylate were the major products. The tri-, tetra-, penta-, and hepta-adenylate were synthesized sequentially, but the levels of the di-adenylate were low throughout the reaction. 2-5 A synthetase was also seen in erythrocytes from specific pathogen-free chickens, suggesting that the enzyme was not produced as a result of microbial infections. 2-5 A synthetases from avian erythrocytes of chicken and pigeon were found not only in cytoplasms, but also in nuclei. No enzyme activity, however, was detected in the nuclear fraction of goose erythrocytes. The molecular size of 2-5 A synthetase in nuclei from chicken erythrocytes was 45, 000-60, 000 daltons, while cytoplasms contained an 85, 000- to 120, 000-dalton enzyme. In addition, the synthetase was present in several types of chicken tissue including liver, intestine, bone marrow, spleen, bursa, pancreas, and thymus, but not in brain, heart, or stomach.View full abstractDownload PDF (596K) -
Shoichi NAKASHIMA, Hiroshi KAMIKAWA1984 Volume 96 Issue 1 Pages 223-228
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy analyzing antibody heterogeneity during the primary immune response to bacterial α-amylase (BαA) in high-responder F1 hybrid mice between C 57 BL/6 (B 6) and C 3 H/He (C 3) mice with the use of isoelectric focusing (IEF), it was shown that the maturation of the primary IgG antibody response consisted of at least two stages. The response of every mouse tested was initiated with the production of specific antibody focused as a limited set of bands in a narrow pH range, and the subsequent rise in antibody titer was associated with the sequential expansion of the spectra involving the appearance of new bands in the pH gradient adjacent to the initial bands. A further rise was accompanied only by intensified staining of the pre-existing bands. These two stages were distinguishable regardless of the antigen dose, although increasing the dose led to widely distributed spectra of focused antibodies and an early shift from the first stage to the second.
The sequential expansion of spectra following the appearance of initial antibodies with limited isoelectric point (pI) values was not unique to the anti-BaA antibody response, because similar results were obtained with the antibody response to an immunologically unrelated antigen, Taka-amylase A (TAA). Thus, the appearance of initial antibodies in a limited pH range, overlapping among all F1 hybrids tested, is not a direct reflection of similarity in the determinant specificities of these antibodies among different mice.View full abstractDownload PDF (1872K) -
Masato UMEDA, Susumu KANDA, Shoshichi NOJIMA, Herbert WIEGANDT, Keizo ...1984 Volume 96 Issue 1 Pages 229-235
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLiposomes could bind and fuse efficiently to human erythrocytes in the presence of HVJ when they contained glycophorin isolated from human erythrocytes (Umeda, M., et al. (1983) J. Biochem. 94, 1955). In the present work we demonstrated that HVJ-induced fusion between liposomes containing glycophorin and erythrocytes was suppressed when GM1 coexisted with glycophorin in the same liposomal membranes. Asialo-GM1 and other gangliosides such as GM3 and sialosylparagloboside did not affect the fusion between the liposomes and erythrocytes. An intermolecular interaction between glycophorin and GM1 was suggested by the ESR spectrum obtained from liposomes containing glycophorin and a ganglioside GM1 analog carrying a nitroxyl spin label in the fatty acyl chains (5 SL-gangliosidoide). The overall splitting value (2 A //) observed in the ESR spectrum of liposomes containing 5 SL-gangliosidoide increased with increase of the amount of glycophorin, whereas 2 A // of spin-labeled phosphatidylcholine was not changed. The increase of 2 A // of 5 SL-gangliosidoide suggests that the mobility of the fatty acyl chain of the gangliosidoide was restricted by the interaction with glycophorin. It can be concluded that GM1 located near glycophorin, a receptor of the virus, interferes with the activity of viral F protein, inhibiting the fusion of liposome to erythrocyte.View full abstractDownload PDF (501K) -
Michiko SEKINE, Toshio ARIGA, Tadashi MIYATAKE, Ryoichi KASE, Akemi SU ...1984 Volume 96 Issue 1 Pages 237-244
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycolipids were isolated from the adrenal glands of seven strains of guinea pig by DEAE-Sephadex and Iatrobeads column chromatographies. The average lipidbound sialic acid content of the adrenal glands was estimated to be 96.0±30.4 nmol/g fresh tissue. The ganglioside fraction contained two major gangliosides which accounted for 82% of the total lipid-bound sialic acid. They were identified as N-acetylneuraminylgalactosylceramide (GM 4) and N-acetylneuraminyllactosylceramide (GM 3). The neutral glycolipid fraction contained one major and three minor glycolipids. The major glycolipid was identified as galactosylceramide, which accounted for 81% of the total glycolipid. The other three glycolipids were identified as Forssman glycolipid (7%), lactosylceramide (5%), and gangliotriaosylceramide (7%). In all strains examined, the glycolipid patterns were similar.View full abstractDownload PDF (2209K) -
II. Binding of Bromophenol BlueMaiyappan SUBRAMANIAN, Bagur S. SHESHADRI, Madhugiri P. VENKATAPPA1984 Volume 96 Issue 1 Pages 245-252
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25°C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques.
Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8°C, also indicate the biphasic nature similar to those observed at 25°C, but with a slight decreased strength of binding.
The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.
Thus, the types of interactions involved in the binding of BPB to lysozyme are mainly electrostatic in nature in addition to hydrogen bonding and van der Waals forces.View full abstractDownload PDF (602K) -
Tangirala RAMAKRISHNA, Madhusudan Waman PANDIT1984 Volume 96 Issue 1 Pages 253-260
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA ribonuclease (RNAase BSD) isolated by us earlier from bovine seminal plasma by DNA-affinity chromatography is shown to be homogeneous on polyacrylamide gel electrophoresis, analytical ultracentrifuge and high performance liquid chromatography. From amino acid analysis, high performance liquid chromatography and enzymatic and physicochemical studies, this enzyme is shown to be identical to RNAase BS-1 reported by D'Alessio et al. ((1972) Eur. J. Biochem. 26, 153-161). Immunological studies support this observation. It has also been shown that, as compared to RNAase A, this enzyme is more sensitive to polyvinyl sulphate inhibition.View full abstractDownload PDF (1805K) -
Noriyuki KASAI, Masaharu NAIKI, Robert K. YU1984 Volume 96 Issue 1 Pages 261-264
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn improved method is described for the detection of ganglioside antigens with the antibodies on thin-layer chromatograms. The method involves the following steps: Separation of gangliosides on a high-performance thin-layer chromatographic plate, application of antibody solution, and detection of bound antibody with [125I] staphylococcal protein A. Using specific antibodies against gangliosides GM 4, GM 1, GD 3, and asialo GM 1 ganglioside, the method allowed positive identification of these antigens on thin-layer plates. It also provides a convenient means of assessing the specificity of an anti-glycolipid antibody.View full abstractDownload PDF (913K) -
Tohru HASEGAWA, Hiroyuki SADANO, Tsuneo OMURA1984 Volume 96 Issue 1 Pages 265-268
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe absorption spectrum of reduced H-450 was reversibly affected by a pH cnange; the Soret peak of the alkaline form was at 448 nm, and that of the acidic form (H-420) at 425 nm. The same spectral change of conversion of reduced H-450 to H-420 was also produced by the action of n-butanol, urea and p-chloromercuribenzoate. These spectral properties of H-450 were similar to those of the conversion of cytochrome P-450 to cytochrome P-420, suggesting the similar heme environments of these two hemoproteins. Reduced H-450 bound carbon monoxide to be transformed into a new spectral species having a Soret peak at 420 nm.View full abstractDownload PDF (280K) -
Toshiaki HIRATSUKA1984 Volume 96 Issue 1 Pages 269-272
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo obtain information about domain-domain contacts in the myosin head, trypsinsplit myosin subfragment-1 (S-1), which mainly consists of 50 K, 26 K, and 20 K fragments, was cross-linked directly by the photodynamic technique under mild conditions (pH 7-8.5, 0°C) using riboflavin 5'-phosphate, protoporphyrin, and methylene blue as sensitizers. Exposure of trypsin split-chymotryptic S-1 to visible light in the presence of the sensitizers resulted in formation of cross-linked products of (20+26) K, (20+50) K, (26+50) K, and (20+26+50) K fragments. The results suggest that three domains in the myosin head are in contact with each other, at least partly, raising the possibility that the communication between the ATP- and actin-binding sites passes through a “short cut, ” i.e., the contact region between the domains.View full abstractDownload PDF (899K) -
Koshin MIHASHI1984 Volume 96 Issue 1 Pages 273-276
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA quasi-steady state rate of actin subunit incorporation into a complex of F-actin, tropomyosin, and troponin was studied in a solution containing MgCl2 0.5mM, ATP 0.2mM, Tris-HCl 5mM (pH 8.0), and either CaCl2 64 μM or EGTA 644 μM at 33°C by adding a small amount (less than 1 μM) of fluorescence-labeled actin monomer. Incorporation of the labeled actin monomer was remarkably much slower in the presence of EGTA, but the rate recovered instantaneously upon addition of an excess amount of CaCl2. The rate was very much reduced by the presence of cytochalasin D (1 μM) and again showed Ca-dependence.View full abstractDownload PDF (239K)
- |<
- <
- 1
- >
- >|