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H Shiokawa

Publications and source records attributed to H Shiokawa.

At least 55 records · Page 3Linked to original sources
PubMed1985

Antigenic determinants of acylphosphatase from porcine skeletal muscle.

Analysis of the quantitative precipitin reaction of acylphosphatase from porcine skeletal muscle with rabbit antiserum indicated the presence of at least two antigenic determinants on the porcine enzyme molecule. Immunological cross-reactivities of acylphosphatases from equine and rabbit skeletal muscles were examined. In double immunodiffusion with the antiserum, the precipitin lines of the porcine and equine enzymes completely fused, while the rabbit enzyme gave no precipitin line. The reaction between the 125I-labeled porcine enzyme and its antibody was inhibited to the same extent by the porcine and equine enzymes, but not by the rabbit enzyme. The three enzymes were similar in net charge and molecular weight on polyacrylamide gel electrophoreses. No conformational difference among the three enzymes was observed in their circular dichroism spectra. The amino acid composition of the rabbit enzyme differed from those of the porcine and equine enzymes in the contents of Glu, Gly, Lys, and Arg. Differences in the sequence of the rabbit enzyme from that of the porcine enzyme were investigated by comparison of the peptide maps of the tryptic peptides of the two enzymes. Four peptides of the rabbit enzyme were located at different positions from those of the porcine enzyme. Three of the four peptides from both enzymes were sequenced and all the tryptic peptides of both enzymes were characterized by amino acid analysis. The tryptic peptides of rabbit enzyme were tentatively aligned on the basis of their amino acid compositions and sequence homologies, compared with the corresponding peptides of the porcine enzyme. Among five amino acid residues of the porcine enzyme, Arg-4, Asp-28, Arg-31, Glu-56, and Ile-68, which are replaced in the rabbit enzyme, Arg-4 and Asp-28 are considered to be included in the antigenic determinants.

Acid Anhydride Hydrolases↗
PubMed1985

Amino acid sequence of acylphosphatase from rabbit skeletal muscle.

The complete amino acid sequence of acylphosphatase from rabbit skeletal muscle has been elucidated by automatic Edman degradation of peptides obtained from staphylococcal protease and trypsin digestions. The enzyme consisted of a single polypeptide chain of 98 amino acid residues, lacking only histidine. Its amino (N)-terminus was blocked by an acetyl group. The presented sequence of rabbit muscle enzyme was compared with those of equine and porcine muscle enzymes. There were four unique replacements, i.e., Arg-4, Asp-28, Arg-31, and Glu-56 in the sequences of both equine and porcine muscle enzymes were replaced by Gly, Gly, Lys, and Asp, respectively, in that of rabbit muscle enzyme. Extensive structural homology was observed among the three enzymes.

Acid Anhydride Hydrolases↗
PubMed1985

Two isozymes of chicken muscle acylphosphatase: purification and properties.

Two acylphosphatases, named Ch1 and Ch2, have been purified from chicken skeletal muscle. The molecular weights were determined to be 11,900 and 12,000 for Ch1 and Ch2, respectively, by sedimentation equilibrium. In the amino acid compositions of Ch1 and Ch2, two residues of histidine were contained in Ch2, but none in Ch1, and one residue of cysteine was contained in Ch1, but none in Ch2. There were 11 lysines and 6 arginines in Ch1, whereas there were 6 lysines and 11 arginines in Ch2. In addition, the contents of methionine, serine, glutamic acid and glutamine, and alanine were considerably different between Ch1 and Ch2. There were also differences in the peptide maps and carboxyl-terminal amino acid sequences (-Ser-Thr-Arg-Tyr-COOH for Ch1, and -Phe-Thr-Ile-Arg-Lys-COOH for Ch2). In the double immunodiffusion, Ch2 did not form a precipitin line with the rabbit anti-Ch1 antiserum. These results indicate that Ch1 and Ch2 are different, genetically specified isozymes of acylphosphatase of chicken skeletal muscle. Ch2 is considered to be a new type of acylphosphatase from skeletal muscle.

Acid Anhydride Hydrolases↗
PubMed1985

Fimbriae from the oral anaerobe Bacteroides gingivalis: physical, chemical, and immunological properties.

Circular dichroism spectra indicated the predominance of beta-sheet structure in Bacteroides gingivalis fimbriae regardless of the presence of sodium dodecyl sulfate. By using a computer program, the alpha-helix, beta-sheet, and beta-turn contents and the remainder were estimated to be 0, 55, 18, and 27%, respectively, judging from the circular dichroism spectra of the fimbriae. Heating for 5 min at 100 degrees C in sodium dodecyl sulfate was necessary to denature the fimbriae into their constituent protein (fimbrilin) monomers with a reduced content of beta-sheet structure. The amino-terminal amino acid sequence of the fimbrilin was different from partial or complete amino acid sequences of fimbrilins so far determined from Bacteroides nodosus, which falls into the same nonfermentative species of the genus Bacteroides as B. gingivalis, and from various other bacteria. Fimbrilin monomers had an isoelectric point of 6.0. Examination of antibodies against fimbriae and sodium dodecyl sulfate-denatured fimbrilin by enzyme-linked immunosorbent assay reinforced a previous notion (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) that different sets of antigenic determinants seemed to be exposed on their surfaces.

Amino Acid Sequence↗
PubMed1984

Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen.

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Crystallization and properties of acylphosphatase from porcine skeletal muscle.

A crystalline acylphosphatase has been obtained from porcine skeletal muscle. The purification procedure consists of extraction with water, phosphocellulose column chromatography, CM-cellulose column chromatography, and crystallization. The enzyme was homogeneous by polyacrylamide gel electrophoresis. A high yield (39%) of the pure enzyme was attained by the use of buffers containing 10 mM 2-mercaptoethanol to prevent dimerization of the enzyme in the purification process. Activity assay in the presence of bovine serum albumin showed a high specific activity of the enzyme (about 7,000 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate). The molecular weight was determined to be 11,100 by sedimentation equilibrium. The amino acid composition of the enzyme was determined. The amino-terminus of the enzyme was blocked and the carboxyl-terminal residue was tyrosine.

Acid Anhydride Hydrolases↗
PubMed1983

Antigenic structure of adenylate kinase from porcine skeletal muscle--II. Immunochemical cross-reactivity of fragments from adenylate kinase.

Specific antibody to a fragment [CBb(2-56)] of porcine muscle adenylate kinase was purified from goat antiserum against adenylate kinase on an immunoadsorbent column. This anti-CBb antibody cross-reacted in solid phase radioimmunoassay with two other CNBr-fragments of adenylate kinase, CBfN(81-125) and CBfC(126-194). This cross-reactivity explained their high inhibition activities in quantitative precipitin reaction between adenylate kinase and goat antiserum shown in the previous work. Cysteinyl residues of the enzyme (positions 25 and 187) were S-cyanylated with 2-nitro-5-thiocyanobenzoic acid and the enzyme was cleaved at these residues. Fragment 1-24 thus obtained was purified. The fragment 1-24, composing the N-terminal half of CBb(2-56), accounted for full activity of CBb to anti-CBb in radioimmunoassay. Hence antigenic region(s) of CBb(2-56) exist in its N-terminal half, 2-24, and this determinant(s) may be closely related to the cross-reactivity among CB-fragments. CBfN also bound to the antibody fraction which had not been adsorbed to CBb-Sepharose (non-anti-CBb). CBfN carried additional antigenic regions. In conclusion, we propose that the antigenic reactive region(s) of adenylate kinase responsible for the cross-reactivity of the CB-fragments are as follows: -Glu-Glu-Lys-Leu-Lys-Lys- (2-7), -Glu-Glu-Phe-Lys-Arg-Lys- (103-108), -Glu-Glu-Thr-Ile-Lys-Lys- (143-148).

Adenylate Kinase↗
PubMed1983

Isolation of three creatine kinases MM from porcine skeletal muscle.

The 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 pI 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.

Amino Acids↗
PubMed1983

Dimer structure of three creatine kinases MM from porcine skeletal muscle.

Three 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 heterodimeric structures (M1M2 and M2M3, respectively), consisting of two nonidentical subunits.

Animals↗
PubMed1983

Properties of three creatine kinases MM from porcine skeletal muscle.

The 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].

Amino Acid Sequence↗
PubMed1982

Antigenic structure of adenylate kinase from porcine skeletal muscle. I. Three immunochemically active peptides obtained by cleavage with cyanogen bromide.

Analysis of the quantitative precipitin reaction of adenylate kinase from porcine skeletal muscle with goat anti-adenylate kinase antiserum indicated that there are at least four antigenic determinants on the enzyme molecule. Porcine adenylate kinase was cleaved with cyanogen bromide, and four peptides were fractionated by ion-exchange chromatographies. Three fragments, CBb (2-56), CBfN (81-125), and CBfC (126-194), inhibited the quantitative precipitin reaction of intact adenylate kinase with goat antiserum. CBb, CBfN, and CBfC also inhibited the binding of 125I-labeled adenylate kinase to the specific antibody purified from goat antiserum. In both inhibition studies, the inhibitory activity of each fragment was extremely high, and reached 70% or more in the latter case. From these results and in view of the presence of the sequence -Glu-Glu-X-X'-Lys (or Arg)-Lys- in each immunochemically active fragment, we suggest that these fragments have similar antigenic determinants which are cross-reactive.

Adenylate Kinase↗
PubMed1981

Carbohydrate-binding activity of concanavalin A containing various numbers of calcium and manganese ions.

After treatment with EDTA, fragment-free concanavalin A (Con A), F3, was fractionated into three major fractions, f1, f2, and f3, by CM-cellulose chromatography. Fully metallized fragment-free Con A, F3M, was prepared by incubation of F3 with excess metal ions and also fractionated into f1M, f2M, and f3M by the same method as for F3. The Con A preparations obtained were analyzed for metal content by atomic absorption spectrophotometry and for number of carbohydrate-binding sites by ultraviolet absorbance change on binding of p-nitrophenyl alpha-D-mannopyranoside (PNP . Man) to Con A. Both f1 (Ca: 0.2, Mn: 0.1) and f1M (Ca: 0.4, Mn: 0.2) had less than 0.6 carbohydrate-binding sites per tetramer of Con A. f2 (Ca: 2.1, Mn: 0.9) and f2M (Ca: 2.1, Mn: 1.9) had 1.9 and 1.8 carbohydrate-binding sites, respectively. f3 (Ca: 4.1, Mn: 2.1) and f3M (Ca: 3.8, Mn: 4.0) had 4.1 and 4.2 carbohydrate-binding sites, respectively. Carbohydrate-binding sites of Con A were in a stoichiometric relation to bound Ca2+. The molar absorptivity change at 317 nm was 2 X 10(3) M-1 . cm-1. Precipitation activity of the above Con A preparations towards glycogen was also estimated using glycogen labeled covalently with Remazolbrilliant Blue R. The precipitation activity of Con A as well as PNP . Man-binding was strongly affected by the number of bound Ca2+. A slight contribution by Mn2+ was also detected in the precipitation activity.

Binding Sites↗
PubMed1981

Crystallization and properties of creatine kinase from equine skeletal muscle.

A crystalline creatine kinase was obtained from equine skeletal muscle. The enzyme was homogeneous, as judged by ultracentrifugation and disc electrophoresis on polyacrylamide gel. The crystalline enzyme had a specific activity of 110 units per mg of protein, that is, 14-fold purification over the crude extract of equine skeletal muscle. The molecular weight of the enzyme was determined to be 84,600 by the conventional low-speed sedimentation equilibrium method, and s020,w was 5.32S. Eight cysteine residues were found on amino acid analysis, two of which were essential for the enzymatic activity.

Amides↗
PubMed1981

Isolation and properties of creatine kinase from porcine skeletal muscle.

Creatine kinase has been isolated in a good yield from porcine skeletal muscle. The enzyme was purified about 8-fold from crude extract of porcine skeletal muscle in a yield of about 44%. The purified enzyme was homogeneous, as judged by sedimentation velocity (S020,W=5.31S), sedimentation equilibrium, and polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 83,200 by high-speed meniscus-depletion sedimentation equilibrium analysis. The molecular weight of its subunit was estimated to be 43,000-44,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the enzyme was determined.

Amino Acids↗