Hiroyuki Shiokawa

Purification and characterization of α1-thiol proteinase inhibitor and its identity with kinin- and fragment 1·2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.

Amino Acid Sequence of Acylphosphatase from Porcine Skeletal Muscle

The amino acid sequence of acylphosphatase from porcine skeletal muscle was determined. It consists of 98 amino acid residues with N-acetylserine at the amino (N)-terminus: Ac-Ser-Thr-Ala-Arg-Pro-Leu-Lys-Ser-Val-Asp-Tyr-Glu-Val-Phe-Gly -Arg-Val-Gln-Gly-Val-Cys-Phe-Arg-Met-Tyr-Thr-Glu-Asp-Glu-Ala-Arg-Lys-Ile -Gly-Val-Val-Gly-Trp-Val-Lys-Asn-Thr-Ser-Lys-Gly-Thr-Val-Thr-Gly-Gln -Val-Gln-Gly-Pro-Glu-Glu-Lys-Val-Asn-Ser-Met-Lys-Ser-Trp-Leu-Ser-Lys -Ile-Gly-Ser-Pro-Ser-Ser-Arg-Ile-Asp-Arg-Thr-Asn-Phe-Ser-Asn-Glu-Lys- Thr-Ile-Ser-Lys-Leu-Glu-Tyr-Ser-Asn-Phe-Ser-Ile-Arg-Tyr-OH. This sequence has three substitutions of amino acid residues, i.e., Thr/Ala, Ile/Val, and Ile/Val at positions 26, 68, and 96, respectively, from that of horse muscle acylphosphatase, formerly the only mammalian acylphosphatase with known sequence.

Distribution and classification of acylphosphatase isozymes.

Distributions of acylphosphatase isozymes among organs of several animal species were investigated. Organ extracts of pig and chicken were treated with isozyme-specific antibodies, subjected to electrophoresis on a polyacrylamide gel, then the gel was stained for acylphosphatase activity. Both animals showed three activity bands; one band was named common type isozyme because of its wide distribution in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte, and the other two bands were named muscle type isozymes because of their localization in skeletal muscle. This classification was supported by selective and quantitative reactions of the isozymes to the isozyme-specific antibodies. Because the two bands of the muscle type have the same amino acid sequence and differ only in modifications on an -SH group, it is suggested that pig and chicken have only the two major types of acylphosphatase. This conclusion was supported by similar experiments on dog, human, rabbit, and pigeon.

Chemical modification of arginine residues of porcine muscle acylphosphatase.

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) from porcine skeletal muscle is inactivated by phenylglyoxal following pseudo-first-order kinetics. The dependence of the apparent first-order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is also first order with respect to the reagent concentration. Among the competitive inhibitors for the enzyme examined, inorganic phosphate and ATP almost completely, and Cl- partially, protect the enzyme against the inactivation. The dissociation constants for inorganic phosphate and ATP determined from protection experiments by these inhibitors agree well with those from inhibition experiments by them. These results support the idea that the modification occurs at the phosphate-binding site. The amino-acid analysis reveals the lack of reaction at residues other than arginine. Circular dichroism spectra of the modified enzymes show that the inactivation seems not to be due to denaturation of the enzyme resulting from the modification of the non-essential arginine residues. The relationship between the loss of the enzyme activity and the number of arginine residues modified in the presence and absence of ATP shows that one arginine residue is possibly responsible for the inactivation of acylphosphatase.

Activity staining of acylphosphatase after gel electrophoresis

Acylphosphatase is an ubiquitous enzyme found in a variety of mammalian and avian tissues. Two isozymes of different amino acid sequence have been found in human, chicken, pig, and horse. To survey the distribution of the acylphosphatase isozymes among animal species and tissues, we have developed an activity staining procedure for the enzyme after electrophoresis on a polyacrylamide gel. Tissue extracts of pigs were subjected to electrophoresis, then the gel was stained for acylphosphatase activity in a solution containing acetyl phosphate and lead nitrate. Three activity bands were observed: the slowest moving one, which coincided with that of purified testis acylphosphatase, was widely distributed in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte; the other two bands, which coincided with monomer and dimer of purified muscle acylphosphatase, were relatively localized in skeletal muscle.

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).

Antigenic structure of adenylate kinase from porcine skeletal muscle. IV. Two antigenic determinants on carboxyl-terminal peptide 126-194.

Delineation of the location(s) of antigenic activity in CNBr peptide 126-194 from porcine skeletal muscle adenylate kinase (AK) was attempted. Peptide 126-194 was digested with chymotrypsin, Staphylococcus aureus V8 protease and trypsin, and several short peptides were purified from the digests by reverse-phase high-performance liquid chromatography (HPLC). Inhibition of the binding of radioiodinated peptide 126-194 to goat antibody to porcine skeletal muscle AK (anti-AK antibody) by the peptides obtained by the enzymatic cleavages was examined by solid phase radioimmunoassay (RIA). At least two antigenic determinants have been identified from the results. One is in the amino (N)-terminal half region 126-154, especially in the vicinity of 131-144, and the other is in the carboxyl (C)-terminal half region 165-183, especially in the vicinity of 165-171. Both of them seem to correspond to exposed and accessible regions in the three-dimensional structure of AK. The correlation between antigenicity and high mobility of the loop in the estimated antigenic region 131-144 is also discussed.

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.