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Volume 93 (1983)
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- Issue 5 Pages 1221-
- Issue 4 Pages 943-
- Issue 3 Pages 669-
- Issue 2 Pages 323-
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Volume 93, Issue 2
Displaying 1-40 of 40 articles from this issue
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Rachid CHRAIBI, Michel WRIGHT1983 Volume 93 Issue 2 Pages 323-330
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThymidine kinase [ATP: thymidine 5'-phosphotransferase, EC 2. 7. 1. 21] has been purified more than 3, 500 fold from microplasmodia of Physarum polycephalum. Properties of the enzyme were determined on preparations purified 1, 400 fold. Thymidine was transformed to dTMP while a stoichiometric quantity of ATP was transformed to ADP. 5-Iododeoxyuridine, 5-bromodeoxyuridine, and 5-fluoro-deoxyuridine acted as competitive inhibitors for the thymidine substrate while 5-bromodeoxyuridine could be used as a substrate. In contrast uridine did not inhibit the enzymatic activity while deoxyuridine was a very poor competitive inhibitor in agreement with the observation that deoxyuridine could not be used as a substrate. Two apparent Michaelis constants were found for thymidine. Only the highest Michaelis constant could be decreased in the presence of increasing concentrations of ATP. Among the various nucleoside mono, di, or triphosphates studied only ATP and to a less extent dATP could be used as phosphate donors. A non competitive inhibition for thymidine was observed with dTTP. dTMP, dTDP, and dTTP acted as competitive inhibitors for ATP. None of the nucleoside mono, di, or triphosphates studied showed an activatory effect at low concentrations of ATP, even in the presence of dTTP. However, dUTP and dGDP acted as competitive inhibitors for ATP.View full abstractDownload PDF (529K) -
Masaru TANOKURA, Yuriko TAWADA, Atsuko ONO, Iwao OHTSUKI1983 Volume 93 Issue 2 Pages 331-337
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14mM Tris/90mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.View full abstractDownload PDF (970K) -
Junko AONO, Sadaaki IWANAGA, Tadao HORIUCHI1983 Volume 93 Issue 2 Pages 339-347
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA tof-like protein that has 434-specific DNA binding activity has been copurified with the 434 tof protein from λimm434cl dv carrier cells. The apparent molecular weight of the new 434-specific DNA binding protein is 9, 000 to 9, 500, a little higher than that of the 434 tof protein, as estimated by SDS gel electrophoresis. Amino acid analysis revealed the protein to be an arginine-rich component whereas the 434 tof protein is a lysine-rich component. The specific binding reaction of the new protein to λimm434 dv DNA is distinct from that of the 434 tof protein in respect to the sigmoid shape of the binding curve and to the temperature dependency. This suggests that the specific binding to λimm434 dv DNA observed with the new protein is due not to a trace of the 434 tof protein contaminating the new protein preparation but rather to the new protein itself. The NH2-terminal 11 residues of the new 434-specific DNA binding protein were sequenced by manual Edman degradation. This technique revealed that the new protein is not a fragment of the 434 tof, cll, or O protein or an NH2-terminal fragment of the cl repressor. The origin and the physiological roles of the new 434-specific DNA binding protein remain unknown.View full abstractDownload PDF (1106K) -
Seikichi TOKU, Yo-ichi NABESHIMA, Kikuo OGATA1983 Volume 93 Issue 2 Pages 349-359
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe long-term effects (up to 12 h) of low dose in vivo actinomycin D treatment, which selectively inhibits rRNA synthesis, on the activity of rat liver for the synthesis of ribosomal proteins relative to that for the synthesis of total protein were investigated. The effects of actinomycin D treatment in vivo and in vitro on the template activity of poly(A)-containing mRNA of rat liver for ribosomal proteins were examined by using a wheat germ cell-free system. The following results were obtained.
1. The activity of rat liver for synthesizing total protein observed in vivo and in vitro was inhibited by actinomycin D treatment even at a small dose.
2. A double-labeling technique using [3H] and [14C]Ieucine in vivo showed that the rate of synthesis of the ribosomal protein fraction relative to that of total protein in actinomycin-treated rat liver (6+6 h) was 1.45 times higher than that in the control rat.
3. By using a wheat germ cell-free system, it was shown that the template activity of poly(A)-containing mRNA for the synthesis of total protein was increased slightly by actinomycin D treatment in vivo. Furthermore, the template activity for the ribosomal protein fraction relative to that for total protein was increased. This increase was observed in most of the ribosomal proteins separated on two-dimen-sional acrylamide gel electrophoresis, although the extents of increase were different among individual ribosomal proteins examined. On the other hand, the selective increase of the template activity for the ribosomal protein fraction was not observed when poly(A)-containing mRNA was incubated with actinomycin D in vitro, although the template activity for total protein was increased slightly.View full abstractDownload PDF (1082K) -
Seikichi TOKU, Yo-ichi NABESHIMA, Kikuo OGATA1983 Volume 93 Issue 2 Pages 361-366
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. When rats received an intraperitoneal injection of [3H]actinomycin D, it bound to the RNA moiety of free and bound polysomes of rat liver. The labeling increased gradually up to 6 h or 6+6h.3 The poly(A)-containing mRNA showed definite radioactivity and its specific activity was higher than that of rRNA, although the total radioactivity of rRNA was markedly higher than that of poly(A)-containing mRNAs.
2. An equilibrium dialysis method using rabbit globin mRNA showed that the binding constant of actinomycin D to globin mRNA was 0.056×106m-1, and globin mRNA had 2 binding sites per mol for actinomycin D.
3. From the results of the present experiments and those described in the preceding paper, it is suggested that one of the mechanisms by which actinomycin D treatment in vivo and in vitro stimulates the template activity of mRNA may be binding to mRNA, which alters the conformation of mRNA.View full abstractDownload PDF (336K) -
Toshihiro HIDAKA, Takashi UETA, Ryohei OGURA1983 Volume 93 Issue 2 Pages 367-373
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIncubation of [1-14C]arachidonic acid (AA) and [1-14C]prostaglandin (PG)H2 with rabbit spleen homogenate and microsomes resulted in the formation of a substance with the chromatographic properties of thromboxane (Tx)B2. The radiolabeled material was indistinguishable from authentic TxB2 on TLC in three solvent systems and on radiometric gas chromatography. The generation of TxB2-like material from AA and PGH2 was not observed after boiling of the homogenate and microsomes, and was completely inhibited by imidazole (5mM). The transformation of AA into the TxB2-like material was not observed during incubation in the presence of indomethacin (28 μm). These results indicate that TxB2 is the principal product of arachidonic acid metabolism by the homogenate or microsomes of rabbit spleen.View full abstractDownload PDF (443K) -
Toshihide TAKASAWA, Hiroyuki SHIOKAWA1983 Volume 93 Issue 2 Pages 375-382
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe purified creatine kinase MM of porcine skeletal muscle [Takasawa, T. & Shiokawa, H. (1981) J. Biochem. 90, 195-204] was separated into three distinct fractions by isoelectric focusing (IEF) in a sucrose gradient column, and the three active fractions were isolated by repeated IEF. There were one major fraction with isoelectric point (pI) 6.57 and two minor fractions with pl 6.74 and pI 6.34, respectively. No differences were observed in the IEF pattern of the enzyme in the presence and absence of dithiothreitol throughout the column. There was no interconversion from one form to another during IEF. The distribution of the three forms on IEF was not affected by adding protease inhibitor to the extraction medium. Of the three fractions, the major fraction had the highest specific activity. The three fractions differed from one another in their amino acid compositions. Not only porcine muscle but also rabbit muscle creatine kinase displayed this type of heterogeneity. Such microheterogeneities may occur widely in muscle creatine kinases.View full abstractDownload PDF (1108K) -
Toshihide TAKASAWA, Hiroyuki SHIOKAWA1983 Volume 93 Issue 2 Pages 383-388
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree active fractions, FI, FII, and FIII, of porcine muscle creatine kinase MM isozyme, which had been isolated as single peaks by repeated isoelectric focusing (IEF), were subjected to reconstitution experiments to study their dimer structure. The specific activities of the three fractions were almost uninfluenced by a dissociation-reassociation procedure. Only one component was observed on IEF after reassociation of the subunits of FII. Three components were observed on IEF after reassociation of subunits of FI and FIII. These results show that FII, with isoelectric point (pI) 6.57, has a homodimeric structure (M2M2), consisting of two identical subunits. FI and FIII, with pI 6.74 and pI 6.34, respectively, have hetero-dimeric structures (M1M2 and M2M3, respectively), consisting of two nonidentical subunits.View full abstractDownload PDF (345K) -
Toshihide TAKASAWA, Masahiko ONODERA, Hiroyuki SHIOKAWA1983 Volume 93 Issue 2 Pages 389-395
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of three fractions (FI, FII, and FIII) of porcine creatine kinase MM, which have been isolated by isoelectric focusing, were compared. Sugars were not detected in them. Their carboxyl-terminal sequences were identical and were determined to be -Thr-Lys by digestion with carboxypeptidases A and B. Immunodiffusion and competitive radioimmunoassay could not differentiate the three fractions from one another. Their amino-terminal sequences revealed that they had different primary structures. At residue 1, although all the three fractions had Pro, FI and FIII had an additional amino acid, Ser. At residue 23, only FI had Leu in addition to Ser, the amino acid common to the three fractions. These results indicate that differences among the three fractions of porcine creatine kinase MM are based on differences in the primary structures of the subunits in their dimer structures, and confirm the conclusion that FII is a homodimer and FI and FIII are heterodimers, which was reported in the preceding paper [Takasawa, T. & Shiokawa, H. (1983) J. Biochem. 93, 383-388].View full abstractDownload PDF (908K) -
Sabu KASAI, Kyoko SUGIMOTO, Retsu MIURA, Toshio YAMANO, Kunio MATSUI1983 Volume 93 Issue 2 Pages 397-402
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS8-Fluoro-8-demethylriboflavin was shown to be a potential active-site-directed reagent for flavoproteins. It reacted with the nucleophiles of N-acetylcysteine (-SH), N-acetyltyrosine (-OH), a-N-acetyllysine, and glycine (ε- and a-NH2, respectively) under fairly mild conditions, and the reaction products were identified. The reactivity of the fluoroflavin was higher than that of 8-chloro-8-demethylribo-flavin, which reacted only with the cysteine derivative among the amino acid derivatives used, and whose pseudo first order rate constant at 23°C was 1/23 of that of the fluoroflavin. The reactivity of the fluoroflavin was also estimated by 13C and 19F NMR spectroscopy. The results showed that this compound is more reactive than the chloroflavin.View full abstractDownload PDF (336K) -
Ryo TAGUCHI, Makoto MIZUNO, Makoto INOUE, Hiroh IKEZAWA1983 Volume 93 Issue 2 Pages 403-412
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (Plase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes.
The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens.
Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin.
Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis.
The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.View full abstractDownload PDF (1301K) -
Ken-ichirou MOROHASHI, Hiroyuki SADANO, Yoshiie OKADA, Tsuneo OMURA1983 Volume 93 Issue 2 Pages 413-419
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hydroxylation of n-hexane by rat liver microsomes was studied and the contribution of different molecular species of cytochrome P-450 to the hydroxylation reaction was examined. In the case of untreated rats, the products of NADPH-dependent n-hexane hydroxylation were 1-, 2-, and 3-hexanols, and the major one was 2-hexanol. Phenobarbital (PB) treatment of animals resulted in a significant increase of the hydroxylation activity. The formation of 2- and 3-hexanols was much more significantly increased than that of 1-hexanol. On the other hand, 3-methylcholanthrene (MC) treatment stimulated the formation of 3-hexanol and the formation of the other two isomeric alcohols was rather decreased. These observations suggested the position specificities of the PB-inducible form (P-450(PB)) and MC-inducible form (P-450(MC)) of cytochrome P-450 in the hydroxylation of n-hexane. Inhibition experiments using antibodies specific to P-450(PB) and P-450(MC) also indicated that P-450(PB) was more active in the hydroxylation at the 2-position whereas P-450(MC) was more specific for the 3-position. NADPH-dependent n-hexane hydroxylation systems were reconstituted by the use of purified NADPH-cytochrome P-450 reductase and P-450(PB) or P-450(MC), and the activities of the reconstituted systems supported the proposed position specificities of these two forms of cytochrome P-450 in n-hexane hydroxylation.View full abstractDownload PDF (421K) -
IV. Assignment of a SubsubgroupFuyuki KAMETANI, Tatsunori TAKAYASU, Satoru SUZUKI, Tomotaka SHINODA, ...1983 Volume 93 Issue 2 Pages 421-429
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid sequence analysis was done on a human J. Bence Jones protein NIG-64 with the major objective of determining the sequence of the variable region. Nineteen tryptic peptides covering 216 residues were isolated from the completely reduced and aminoethylated protein, and 17 of these were completely sequenced. These comprised the entire variable region and 11 from the constant region. For the remaining peptides covering the rest of the constant region, only partial sequences or the amino acid compositions were determined. All the tryptic peptides could be arranged in order on the basis of the above results and homology with other human λ light chains of the same isotype. The sequence of the variable region of the protein is highly homologous with that of protein New of subgroup VλI as compared with other proteins of the same subgroup, suggesting that subgroup VλI may be further divided into subsubgroups, namely subsubgroups VλI-1 and VλI-2.View full abstractDownload PDF (471K) -
Kiyoshi IMAI, Tokiko HAMA, Takeshi KATO1983 Volume 93 Issue 2 Pages 431-437
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDipeptidyl aminopeptidase, which hydrolyzes the 7-(Gly-Pro)-4-methylcoumarinamide, has been purified from the brains of 3 week-old rats. It was purified about 2, 600-fold by column chromatography on CM-cellulose, hydroxyapatite and Gly-Pro AH-Sepharose. This enzyme hydrolyzed Lys-Ala-β-naphthylamide well with an optimum pH of 5.5. It was inhibited by diisopropyl fluorophosphate, phenyl-methanesulfonyl fluoride, some cations, and puromycin, but was not inhibited by p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, EDTA, iodoacetic acid, and bacitracin, indicating that rat brain dipeptidyl aminopeptidase is a serine protease. This enzyme showed a molecular weight of 220, 000 by gel filtration and of 51, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The properties of purified rat brain dipeptidyl aminopeptidase were similar to those of bovine pituitary dipeptidyl peptidase II, but the molecular weight and substrate specificity of these enzymes were different.View full abstractDownload PDF (887K) -
Shoko MIYAZAWA, Hisashi OZASA, Shuichi FURUTA, Takashi OSUMI, Takashi ...1983 Volume 93 Issue 2 Pages 439-451
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCarnitine acetyltransferase was purified from rat liver after induction of the enzyme by feeding with di(2-ethylhexyl)phthalate. Two enzyme sources were used: the mitochondrial fraction and the homogenate of the liver. The purification procedure was essentially the same for the two enzyme sources. The enzyme purified from the mitochondrial fraction consisted of two different polypeptides with molecular weights of 36, 500 and 27, 000, whereas that from the homogenate consisted of one polypeptide with a molecular weight of 67, 500. Amino acid compositions and peptide maps of the limited proteolytic products of the two enzyme preparations were nearly the same. Their antibodies were cross-reactive. Catalytic properties of the two preparations were nearly the same: the specific enzyme activities, double reciprocal plots of initial velocity study, substrate specificities for aeylcarnitines having various carbon chain lengths, apparent Michaelis constants for substrates. On electrophoresis of the immunoprecipitate obtained after incubation of the mito-chondrial extract, the two immunoreactive polypeptides with molecular weights of 36, 500 and 27, 000 were found. But only one polypeptide, with molecular weight of 67, 500, was detected when the protease inhibitors were added to the mitochondrial extract. It was concluded that the enzyme in the mitochondrial fraction was a monomeric form but was converted into a dimeric form by proteolytic modification after the disruption of mitochondria. The preparation from the post-mito-chondrial fraction, which had a lower specific activity, contained two polypeptides whose molecular weights were 69, 000 and 67, 500. They could not be separated from each other throughout the purification. The peptide maps of the products of the limited proteolysis were very similar.View full abstractDownload PDF (4313K) -
Shoko MIYAZAWA, Hisashi OZASA, Shuichi FURUTA, Takashi OSUMI, Takashi ...1983 Volume 93 Issue 2 Pages 453-459
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMale Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4, 5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocyte-lysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw=69, 000) larger than the mature enzyme (Mw=67, 500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.View full abstractDownload PDF (1709K) -
Tamio HIRABAYASHI, Ryoji TAMURA, Ikuo MITSUI, Yoshio WATANABE1983 Volume 93 Issue 2 Pages 461-468
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTotal protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents. In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s. It was eventually found that employment of 8M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure.
A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCI extract gave a ‘protein map’ on which most proteins were located in their respective positions, including proteins with more than 200, 000 mol. wt. Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahyntena actin may have characteristics far different from those of skeletal muscle actin.View full abstractDownload PDF (2687K) -
Tadashi YOSHIMOTO, Toshiko SAEKI, Daisuke TSURU1983 Volume 93 Issue 2 Pages 469-477
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProline iminopeptidase [EC 3. 4. 11. 5] was purified about 1, 700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-β-naph-thylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X=amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulf-hydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58, 000 by gel filtration on Toyopearl and 60, 000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.View full abstractDownload PDF (1223K) -
Shigemi NORIOKA, Tokuji IKENAKA1983 Volume 93 Issue 2 Pages 479-485
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of peanut trypsin-chymotrypsin inhibitor, B-111, was determined by conventional methods. The limited proteolysis of B-Ill with trypsin indicated the reactive sites of B-III for trypsin to be Arg(10)-Arg(11) and Arg(38)-Ser(39). Comparison of the established sequence of B-III with those of other Bowman-Birk type double-headed protease inhibitors indicated that B-Ill has four amino acid insertions and one amino acid deletion. It is especially interesting that the amino acid residue in the P1' position (No. 11) of the first reactive site for trypsin is arginine instead of serine, which seems to be conserved at the P, ' position of the reactive sites of all Bowman-Birk type protease inhibitors.View full abstractDownload PDF (370K) -
Kenzo CHIBA, Takao OHYASHIKI, Tetsuro MOHRI1983 Volume 93 Issue 2 Pages 487-493
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn equilibrium dialysis study has revealed that porcine intestinal calcium-binding protein (CaBP) has two binding sites for Ca2+ whose dissociation constants (Kd) are the same, 0.56 μM. The intrinsic fluorescence spectrum of the CaBP shows a peak (at 303 nm) in tyrosine band. Ca2+ binding to the CaBP induces a monophasic increase in the intensity of intrinsic fluorescence without any shift to either excitation or emission maximum. The change in the fluorescence intensity induced by Ca2+ binding is complete at a bound Ca2+/CaBP molar ratio of about 2, and the apparent Kd value is 0.51 μM. The same bound Ca2+/CaBP molar ratio has been obtained from the maximal changes in UV absorption and CD spectrum of the CaBP upon addition of Ca2+. A CD study has shown an about 5% increase in α-helix content in the CaBP at the maximal binding of Ca2+. All these results indicate that the porcine intestinal CaBP has two high-affinity binding sites with equal affinity for Ca2+ and suggest that it undergoes a quantitative conformation change accompany-ing Ca2+ binding in the vicinity of single tyrosine and phenylalanine residues of the protein molecule.View full abstractDownload PDF (350K) -
Eiichi SAITOH, Satoko ISEMURA, Kazuo SANADA1983 Volume 93 Issue 2 Pages 495-502
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of a basic proline-rich peptide, P-D, isolated from human parotid saliva was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Glu-Gly-Asn-Lys-Pro-GIn-GIy-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Pro-GIy-Gly-Asn-Pro-GIn-GIn-Pro-GIn-AIa-Pro-Pro-Ala-GIy-Lys-Pro-GIn-Gly-Pro-Pro-Pro-Pro-Pro-GIn-GIy-GIy-Arg-Pro-Pro-Arg-Pro-AIa-GIn-Gly-Gln-GIn-Pro-Pro-Gln by conventional methods. P-D contains a number of repeating sequences and shows a high degree of homology with the salivary basic proline-rich peptides P-C and P-E.View full abstractDownload PDF (480K) -
Nobuko WATANABE, Morio UMENO, Motoaki ANAL1983 Volume 93 Issue 2 Pages 503-511
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography. ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography. Each peak fraction was further purified by ATP-agarose affinity chromatography. Peak A and Peak B were eluted from an ATP-agarose column at 0.14M and 0.28M KCl, respectively, each as a single peak. Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA. The two peaks are optimally active at 69°C and have similar optimal pH ranges from 8.2 to 9.2. The two purified peaks were unstable on storage at -20°C, but were remarkably stabilized by addition of 0.4mg/ml bovine serum albumin. Ammonium sulfate strongly inhibits the activities of both peaks. The molecular weights of Peak A and Peak B are about 170, 000 as estimated by glycerol gradient sedimentation. The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups. The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments. The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.View full abstractDownload PDF (1024K) -
Yoshiki OHBA, Koji TOYODA1983 Volume 93 Issue 2 Pages 513-523
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNuclei were isolated from mouse lymphoma L5178Y cells in the exponential growth phase, and chromatin was prepared by mechanical treatment of the nuclei. The nuclei and the chromatin were then digested to various extents with micrococcal nuclease and the resulting mono- and dinucleosome fractions of the two preparations were compared.
During progressive digestion mononucleosomes from chromatin retained H1 histone and a DNA length of 165 base pairs, whereas those from nuclei released H1 histone and the length of their DNA decreased to 140 base pairs at an early stage of digestion.
These nucleosomal preparations were always associated with nonhistone proteins. The dinucleosomes from nuclei contained larger amounts of nonhistone proteins than those from chromatin, but half of these proteins was released during the process of cleavage into mononucleosomes. The final mononucleosome preparation from nuclei retained 20% less nonhistone proteins than that from chromatin. The contents of nonhistone proteins in mono- and dinucleosomes from chromatin were the same. The electrophoretical distributions of molecular species of nonhistone proteins in mononucleosomes from nuclei and chromatin were different from each other: during digestion the profile of the former changed, whereas that of the latter remained constant.
It is tentatively concluded that both HI histone and nonhistone proteins were bound to nucleosomes more or less loosely in intact nuclei in situ, but that when the nuclear structure was disrupted these proteins became more tightly bound.View full abstractDownload PDF (1205K) -
Kohei SAWAKI, Ryo TAGUCHI, Hiroh IKEZAWA1983 Volume 93 Issue 2 Pages 525-535
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents.
In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used.
On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phos-phatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed micro-somes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate).
When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5mM) of substrate, glucose-6-P.
The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phos-phatidylethanolamine, or phosphatidylinositol.
The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.View full abstractDownload PDF (687K) -
Kohei SAWAKI, Ryo TAGUCHI, Hiroh IKEZAWA1983 Volume 93 Issue 2 Pages 537-546
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThrough kinetic analysis, the relationships between the glucose-6-phosphatase system and constituent phospholipids were studied in rat liver microsomes.
When phosphoglycerides such as phosphatidylcholine and phosphatidylethanol-amine on the microsomal membrane were hydrolyzed by phospholipase C of C. perfringens, the activities of glucose-6-P phosphohydrolase and glucose-6-P:glucose phosphotransferase both decreased with or without subsequent exposure to taurocholate. In these cases, the Michaelis constants (Km) for glucose-6-P were increased, concomitant with the decrease in the maximal velocities (Vmax) for glucose-6-P hydrolysis. On exposure to taurocholate, the apparent Km for glucose of phos-photransferase was decreased.
When phosphatidylinositol was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the activities of phosphohydrolase and phos-photransferase were both decreased on exposure to taurocholate. In this case, the value of Vmax of phosphohydrolase was decreased and that of Km for glucose-6-P was slightly decreased, while the apparent Km for glucose of phosphotransferase was increased. Without exposure to detergent, the activities of phosphohydrolase and phosphotransferase both decreased at glucose-6-P concentrations higher than 10mM. However, at a concentration lower than I mm, the activity of phospho-hydrolase became higher than that of the control, and Vmax and Km for glucose-6-P were decreased.
A similar tendency was also observed in microsomes where membranous phos-phatidylinositol was hydrolyzed, when they were treated with DIDS (an anion-transport inhibitor).
From these results, it is concluded that the activity of glucose-6-phosphatase is greatly influenced by changes of the phospholipids on the microsomal membrane, and the activity of glucose-6-P translocase is stimulated by the breakdown of phos-phatidylinositol.View full abstractDownload PDF (525K) -
Yukihide OHSUMI, Toyoko ISHIKAWA, Keitaro KATO1983 Volume 93 Issue 2 Pages 547-556
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rapid and simplified method for the preparation of lysosomal membranes from rat livers was developed. Enzymic characterization showed that the lysosomal membrane preparation isolated by this procedure was almost free from mitochondria, peroxisomes, and endoplasmic reticulum. Acid phosphatase was used as a marker enzyme for lysosomal membrane, because about 40% of the total acid phosphatase activity in the lysosomes was associated with the membranes. With this method, the yield of the purified membrane was 115 μg/g wet weight of liver and the relative specific activity of acid phosphatase in the purified membrane was 105-110. On SDS-polyacrylamide gel electrophoretic analysis, the purified membrane revealed glycoprotein bands in the region of M. W. between 60, 000 and 110, 000, which were characteristic of the tritosomal membrane. Our lysosomal membrane preparation was compared with lysosomal membranes prepared from normal rat liver lysosomes isolated by the method of Wattiaux et al. [(1978) J. Cell Biol. 78, 349-368]. Although the specific activity of acid phosphatase in our membrane preparation was higher than that in the membrane prepared by the existing method, the electrophoretic profiles of both membrane preparations were quite similar.View full abstractDownload PDF (2281K) -
Kiyofumi MARUYAMA1983 Volume 93 Issue 2 Pages 557-565
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA hydrolase which catalyzes specifically the interconversion between 2-pyrone-4, 6-dicarboxylate and 4-oxalmesaconate was purified about 410-fold with a 16% yield from cell-free extracts of Pseudomonas ochraceae grown with phthalate. Upon disc gel electrophoresis, the enzyme preparation gave a single band which was coincident with the enzyme activity. The molecular weight of the enzyme was estimated to be 31, 000 by gel filtration on Sephadex G-75 and 33, 000 by sodium dodecyl sulfate gel electrophoresis. The isoelectric point of the enzyme was determined to be at pH 5.49 by isoelectric focusing. The enzyme is specific for 2-pyrone-4, 6-dicarbox-ylate, and various other lactones did not serve as substrates. The stoichiometry of 2-pyrone-4, 6-dicarboxylate hydrolysis, 4-oxalmesaconate formation and proton production was approximately 1:1:1. The optimum pHs are 8.5 and 6.0 for hydrolysis and synthesis of 2-pyrone-4, 6-dicarboxylate, respectively. Km values are 87 and 26 μM for 2-pyrone-4, 6-dicarboxylate and 4-oxalmesaconate, respectively. At pH 8.5, the ratio of 4-oxalmesaconate to 2-pyrone-4, 6-dicarboxylate at equilibrium is about 2.2. Thiol reagents such as HgCI_ and p-chloromercuribenzoate strongly inhibit the enzyme activity.View full abstractDownload PDF (1103K) -
Kiyofumi MARUYAMA1983 Volume 93 Issue 2 Pages 567-574
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enzyme responsible for the degradation of 4-oxalmesaconate was partially purified from Pseudomonas ochraceae grown with phthalate. Column chromatography on DEAE-cellulose caused separation into two distinct enzymes, I and II. 4-Oxalmesaconate was converted into pyruvate and oxalacetate in the presence of MgCl2 and enzymes I and II. Optimum pH of the reaction was observed at pH 8.2 in Tris-HCl buffer. MgCl2 could be replaced by MnCl2 or CoCl2 Both enzymes were stable to heat-treatment at 65°C for 10min. Analyses of time course, products and substrate specificity of the enzyme reaction accounted for the functions of two enzymes. Enzyme I (molecular weight 55, 000, isoelectric point 5.1) hydrated 4-oxalmesaconate to give 4-oxalcitramate and may be classified as a hydro-lyase. Enzyme II (160, 000, 5.0) catalyzed the aldolitic cleavage of 4-oxalcitramalate to pyruvate and oxalacetate in the presence of MgCl2. Enzyme II also cleaved 4-hydroxy-4-methyl-2-oxoglutarate into pyruvate. Stoichiometry of the enzyme reaction suggested that enzyme II-catalyzed cleavage occurred on only one enantiomer of the substrates. Furthermore, the metabolic pathway for the dissimilation of protocatechuate in P. ochraceae is presented and discussed in comparison with the pathway postulated previously by other workers.View full abstractDownload PDF (465K) -
Hiromichi OKUDA, Takahiro TSUJITA, Michihiro SUMIDA, Yukari TAKAHASHI, ...1983 Volume 93 Issue 2 Pages 575-582
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndogenous lipid droplets were prepared by subjecting fat cells to hypotonic shock and to Triton X-100 treatment. The structure of the endogenous lipid droplet fraction was examined by scanning and transmission electron microscopies. Neither intact fat cells nor disrupted cell membranes were detectable in the endogenous lipid droplet fraction. With this endogenous substrate, epinephrine elicited lipolysis with either hormone-sensitive lipase or lipoprotein lipase, but no cyclic AMP-protein kinase mediated stimulation of lipolysis was observed. On the other hand, epinephrine did not stimulate lipolysis when triolein emulsified with arabic gum was used as substrate. With the latter exogenous substrate, however, cyclic AMP-protein kinase was found to stimulate lipolysis with hormone-sensitive lipase as enzyme. These results agree with the proposal of Wise and Jungas that the epine-phrine-stimulated increase of hydrolysis of endogenous fat is not mediated through cyclic AMP-protein kinase. A possible mechanism of hydrolysis of endogenous fat by induction of lipolysis by epinephrine in fat cells is discussed.View full abstractDownload PDF (1219K) -
Toshiharu SUZUKI, Shunji NATORI1983 Volume 93 Issue 2 Pages 583-590
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHemolymph of Bombyx mori larvae was found to contain activity to agglutinate trypsinized and glutaraldehyde-fixed sheep red blood cells (fixed SRBC). The specific activity of the hemagglutinin changed during development from the fifth instar to the adult with a transient increase just before pupation. This activity was specifically inhibited by glucuronic acid and heparin. The material with hemagglutinating activity was partially purified by gel filtration on Sephacryl S-300. A protein with molecular weight of 260, 000 that preferentially binds to fixed SRBC was identified using radioiodinated hemagglutinin. Antibody raised against this protein specifically inhibited the activity of partially purified hemagglutinin, indicating that this protein is essential for hemagglutination.View full abstractDownload PDF (2176K) -
Teruko TAKANO, Masaharu TAKIGAWA, Fujio SUZUKI1983 Volume 93 Issue 2 Pages 591-598
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, it was demonstrated that parathyroid hormone (PTH) induces a series of events, successive increases of the level of cyclic AMP, the activity of ornithine decarboxylase [ODC; EC 4. 1. 1. 17] and glycosaminoglycan synthesis, a characteristic chondrocyte phenotype, in rabbit costal chondrocytes in culture. Cyclic AMP analogues also increase ODC activity and glycosaminoglycan synthesis in these cells. Therefore, in the present work, the role of polyamines in expression of the differentiated phenotype of chondrocytes was investigated. DL-α-Hydrazino-δ-aminovaleric acid (DL-HAVA), a potent inhibitor of ODC, inhibited the increase in polyamine levels of quiescent cultures of chondrocytes treated with PTH. DL-HAVA also inhibited stimulation of expression of the differentiated phenotype of chondrocytes by PTH, as judged by decreases in glycosaminoglycan synthesis and metachromasia on toluidine blue staining. Similarly, DL-HAVA inhibited stimulation of expression of the differentiated phenotype of chondrocytes by dibutyryl cyclic AMP. The inhibitory effect on expression of the differentiated phenotype of chondrocytes by DL-HAVA was effectively diminished by addition of polyamines. Under the same conditions, PTH, DL-HAVA, or polyamines had little effect on DNA synthesis. These findings suggest that the rise in polyamine levels induced by PTH, which is mediated by an increase of cyclic AMP concentration, is essential for expression of the differentiated phenotype of chondrocytes.View full abstractDownload PDF (1096K) -
Toshihiro TSUDZUKI1983 Volume 93 Issue 2 Pages 599-606
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of [3H]dopamine with acidic proteins present in the synaptosomal cytoplasm of rat brain was studied from the point of view of Ca2+-dependent release of dopamine as a neurotransmitter. Some of the proteins were glycoproteins and showed various affinities for [3H]dopamine. [3H]Dopamine bound to an acidic protein with a molecular weight of about 45, 000 daltons became reversibly unbound in the presence of Ca2+, while that bound to other proteins did not. The amount of [3H]dopamine bound to the 45, 000-dalton glycoprotein was 48.7 pmol/mg protein, and the Ca2+-concentration required to release half of the bound dopamine was found to be 0.2 to 0.25mM under the experimental conditions. However, Cal2+ did not bind to any component of the acidic proteins and it is likely that the cation dissociates the bound dopamine by forming a complex with it. No calmodulin (-like protein) was detectable among the acidic proteins. Possible roles of the glycoproteins are discussed in relation to a Ca2+-dependent mechanism of neuro-transmitter release.View full abstractDownload PDF (1783K) -
Takao OJIMA, Kiyoyoshi NISHITA, Shizuo WATANABE1983 Volume 93 Issue 2 Pages 607-613
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn a previous report (J. Biochem. 89, 1333-1335, 1981) we showed that 30°C-treat-ment (pCa<6 and 2mM MgCl2), like EDTA-treatment, caused a reversible removal of regulatory light-chains from Akazara adductor myosin. Utilizing the heat-treat-ment, we now show (a) that not half but the total removal of regulatory light-chains from Akazara myosin is required for a complete loss of calcium sensitivity of myo-sin-ATPase, and (b) that recombination of not 1 but 2 mol of regulatory light-chains is required for a full recovery of calcium sensitivity of both myosin-ATPase and actomyosin-superprecipitation. These (a, b) are what we showed previously with EDTA-treatment (J. Biochem. 85, 1543-1546, 1979 and 86, 663-673, 1979), thus establishing that in all respects we tested, the heat-treatment is as good as EDTA-treatment for reversible removal of regulatory light-chains.
We also show that the presence of actin during heat-treatment of myosin prevented regulatory light-chains from being released (at pCa 7) and that how well the release was prevented depended on the MgCI2 concentration during the heat-treatment.View full abstractDownload PDF (1652K) -
Shigetaka KITAJIMA, Shigeki MINAKAMI1983 Volume 93 Issue 2 Pages 615-620
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36, 000 and 32, 000 daltons, respectively, which are comparable to those of the deter-gent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver micro-somes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.View full abstractDownload PDF (2051K) -
Kimiko MURAKAMI-MUROFUSHI, Keiko TADANO, Ineo ISHIZUKA1983 Volume 93 Issue 2 Pages 621-629
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel trisialosyl ganglioside was isolated from hog kidney cortex and purified in a homogeneous form with a yield of 2.5 nmol/g wet tissue or 4.9 mol% of the total gangliosides. The pure substance was characterized chemically and enzymatically, and the results suggested the structure of this ganglioside is NeuAca2-8NeuAcα2-8NeuAcα2-3Galβ1-4G1eNAcβ1-3Galp1-4Glcβ1-1Cer (trisialosyl lactoneotetraosyl-ceramide, LT1, IV3α(NeuAc)3nLcOse4Cer).View full abstractDownload PDF (1835K) -
Yasuhiro NATORI, Ken KARASAWA, Hiroyuki ARAI, Yumiko TAMORI-NATORI, Sh ...1983 Volume 93 Issue 2 Pages 631-637
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhospholipase A2 of rat liver mitochondria was purified approximately 1, 400-fold by extraction with KCl, and chromatographies on a Sephadex G-75 column and a diacyl-glycerophosphocholine-Sepharose affinity column. The purified enzyme was very labile when incubated either at 37°C or 0°C, and lost its activity within a few hours. Phospholipids or detergents in the solution protected the enzyme against inactivation. The purified phospholipase A2 preferentially hydrolyzed phospha-tidylethanolamine, especially if it contained linoleic acid. The enzyme showed low activity for phosphatidylcholine or phosphatidylinositol.View full abstractDownload PDF (464K) -
Hiromu MUROFUSHI, Yasufumi MINAMI, Gen MATSUMOTO, Hikoichi SAKAI1983 Volume 93 Issue 2 Pages 639-650
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA high molecular weight protein has been partially purified from sheaths of squid giant axons. This protein fraction was capable of restoring the membrane excitability of the squid axon which had been destroyed by internal perfusion of microtubule poison, when perfused along with microtubule proteins (Matsumoto et al. (1979) J. Biochem. 86, 1155-1158).
This protein, designated as 260 K protein, was purified by gel filtration and Con A-Sepharose affinity chromatography. The apparent molecular weight of the axonal protein was estimated to be 260, 000 by electrophoresis in the presence of sodium dodecylsulfate. This protein was revealed to be a glycoprotein.
When phosphocellulose-purified tubulin was incubated with 260 K protein at 36°C in the presence of dimethylsulfoxide, turbidity of the solution was much increased. 260 K protein co-sedimented with microtubules assembled from purified tubulin. Light microscopic and electron microscopic observations revealed that the high turbidity was due to bundling of microtubules which was caused by 260 K protein.
On the other hand, the effect of this protein on the turbidity increase was not so prominent when microtubules were assembled from microtubule proteins consisting of tubulin and microtubule-associated proteins.
High shear and low shear viscometry and co-sedimentation experiments revealed that 260 K protein had little effect on actin polymerization under the same medium conditions as used in tubulin polymerization.View full abstractDownload PDF (2695K) -
Hiroaki NAKAJIMA, Akira KUROSAKA, Atsuko FUJISAWA (nee SEHARA), Toshis ...1983 Volume 93 Issue 2 Pages 651-659
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mucin-type glycoprotein was isolated from a human rectal adenocarcinoma, mainly by gel filtration and hydroxyapatite treatment. The glycoprotein, designated as rectal mucin-type glycoprotein (RMG), was great in amount, accounting for about 1% of the wet tissue weight. From a non-cancerous area of the patient's intestine, a similar glycoprotein reacting with anti-RMG antibodies was obtained, but the tissue content was less than 10% of the RMG content.
Purified RMG contained about 70% carbohydrate in mass, and is composed of about equimolar amounts of sialic acid, galactose, N-acetylglucosamine and N-acetylgalactosamine. The polypeptide core was characterized by high contents of threonine, serine, and proline. A marked difference between RMG and the normal glycoproteins was that the sialic acid content was much higher in RMG.
Of the total N-acetylgalactosamine convertible to N-acetylgalactosaminitol by reductive cleavage with alkaline borohydride, about 15% was free and the rest occupied the reducing ends of acidic oligosaccharides. The acidic oligosaccharides were fractionated into a fraction of high molecular weight and a series of oligosac-charides in which di- and trisaccharides containing sialic acid were dominant. The high molecular weight fraction contained esterified sulfate.View full abstractDownload PDF (1214K) -
Akira MURASUGI, Chiaki WADA, Yukimasa HAYASHI1983 Volume 93 Issue 2 Pages 661-664
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (1). It was also reported that CdBPI and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2). Now, we have found that Cd-BP1 contains about 1 mol of acidlabile sulfide per mol, and Cd-BP2 contains no labile sulfide. The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BPI. Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BPI is the first instance in this field.View full abstractDownload PDF (253K) -
Ikuo ASHIKAWA, Kazuhiko KINOSITA Jr., Akira IKEGAMI, Yoshifumi NISHIMU ...1983 Volume 93 Issue 2 Pages 665-668
Published: February 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have studied the dynamics of DNA in nucleosome core particles and in the linker region of chromatin using nanosecond fluorescence anisotropy decay measurements of intercalated ethidium. DNA in the core undergoes torsional motions to the same extent as the linker DNA in extended chromatin. We therefore concluded that the binding of DNA to the histone octamer is relatively weak or limited to a few points; stretches of at least several tens of base pairs exist which can move as freely as DNA in solution.View full abstractDownload PDF (224K)
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