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.     

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.     

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.     

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.     

Duck skeletal muscle acylphosphatase: Primary structure

The complete amino acid sequence of duck skeletal muscle acylphosphatase is presented. The sequence was studied by the manual Edman degradation of the complete series of tryptic peptides and the amino acid composition of peptic peptides. The NH₂-terminus is acetylated, and the polypeptide consists of 102 amino acid residues. The sequence is compared with other known acylphosphatases from the skeletal muscle of several vertebrate species.     

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.     

The complete amino acid sequence of bovine skeletal muscle acylphosphatase

The primary structure of bovine skeletal muscle acylphosphatase was determined by performing the sequence analyses of the complete series of tryptic peptides. The amino acid composition of the entire series of peptic peptides was used to reconstruct the sequence by the overlapping method. The proposed structure is further confirmed by analogy with known amino acid sequences of acylphosphatase from skeletal muscle of other vertebrate species. The length of the polypeptide chain is 98 residues, identical to the length of the enzymes from other known mammalian species, but different from that found in turkey. The enzyme is NH2-acetylated and a comparison with the analogous molecular forms from other vertebrate species indicates that there are several long polypeptide stretches strictly conserved (93-97% identical position among mammals, and about 80% between calf and turkey enzymes).     

Components of purified sarcolemma from porcine skeletal muscle

Sarcolemmal membranes were isolated from porcine skeletal muscle by modifications of a LiBr-extraction technique. Latency determinations of acetylcholinesterase, ouabain-sensitive p-nitrophenylphosphatase, [3H]ouabain binding, and (Na+ + K+)-ATPase activities indicated that 65-76% of the membranes were sealed inside-out vesicles. The preparations were enriched in cholesterol and phospholipid, and demonstrated adenylate cyclase activity and both cAMP and cGMP phosphodiesterase activities. An indication of the purity of this fraction was that the Ca2+-ATPase activity (0.13 mumol Pi mg-1 min-1 at 37 degrees C) was 3.8% of that of porcine skeletal muscle sarcoplasmic reticulum preparations. Pertussis toxin specifically catalyzed the ADP-ribosylation of a Mr 41,000 sarcolemmal protein, indicating the presence of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, Ni. An endogenous ADP-ribosyltransferase activity, with several membrane protein substrates, was also demonstrated. The addition of exogenous cAMP-dependent protein kinase or calmodulin promoted the phosphorylation of a number of sarcolemmal proteins. The calmodulin-dependent phosphorylation exhibited an approximate K 1/2 for Ca2+ of 0.5 microM, and an approximate K 1/2 for calmodulin of 0.1 microM. 125I-Calmodulin affinity labeling of the sarcolemma, using dithiobis(succinimidyl propionate), demonstrated the presence of Mr 160,000 and 280,000 calmodulin-binding components in these membranes. These results demonstrate that this porcine preparation will be valuable in the study of skeletal muscle sarcolemmal ion transport, protein and hormonal receptors, and protein kinase-catalyzed phosphorylation.     

Secondary structure of acylphosphatase from rabbit skeletal muscle. A nuclear magnetic resonance study

Sequence-specific assignment of 1H nuclear magnetic resonance spectra of acylphosphatase (EC 3.6.1.7) isolated from rabbit skeletal muscle have made it possible to identify short distance constraints from nuclear Overhauser enhancement spectra, to evaluate spin-spin coupling constants of many backbone amide hydrogens and to assess their slow exchange with deuterons in 2H2O solution. Analysis of these data show that the major regular secondary structure of the enzyme consists of five extended beta-strands, four of which are arranged in an antiparallel beta-sheet, while the fifth is attached parallel. A helix consisting of 11 residues has also been identified. Consideration of additional distance constraints between sequentially remote residues has allowed us to give an outline of the overall fold of the protein.     

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.     

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].     

Rabbit skeletal muscle acylphosphatase: the amino acid sequence of form Ra1

Acylphosphatase was purified from rabbit skeletal muscle by a procedure involving an affinity chromatography step on immunoadsorbent and subsequent ion-exchange chromatography. Three molecular forms with acylphosphatase activity, named Ra1, Ra2, and Ra3, were purified and characterized with respect to molecular weight, amino acid composition, and main kinetic parameters. The amino acid sequence of Ra1 is given in the present paper. The Ra1 form consists of a single polypeptide chain of 98 amino acid residues and contains only one cysteine residue at position 21 that is S-S bound to glutathione. The polypeptide chain has an acetyl group blocking the NH2 terminus. Ra1, Ra2, and Ra3 are compared with the corresponding molecular forms isolated from skeletal muscle of horse and turkey.     

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.     

Purification and properties of porcine testis acylphosphatase

Acylphosphatase has been purified from porcine testis and its properties were compared with those of porcine skeletal muscle acylphosphatase. The molecular weight of the testis enzyme was found to be 10,600, similar to that of porcine skeletal muscle acylphosphatase, on sedimentation equilibrium analysis. The specific activity of the testis enzyme was 10,800 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate, i.e., higher than that of the muscle enzyme, 7,200 mumol/min/mg, under the same conditions. The pI of the testis enzyme was 8.3, i.e., lower than that of the muscle enzyme, 10.6. There were marked differences in the amino acid compositions of the two enzymes. In particular two histidine residues were present in the testis enzyme but none were present in the muscle enzyme, and no cysteine residue was present in the testis enzyme but one was present in the muscle enzyme. The carboxyl terminal amino acid of the testis enzyme seemed to be lysine, while that of the muscle enzyme is tyrosine. The peptide maps of the testis and muscle enzymes indicated considerable differences in the amino acid sequences of the two enzymes. Differences in the antigenic structures of the two enzymes were demonstrated on enzyme linked immunoassaying and double immunodiffusion. These results indicate that the porcine testis acylphosphatase is an isozyme different from the porcine skeletal muscle acylphosphatase.     

Purification and properties of aminopeptidase C from porcine skeletal muscle.

1. Aminopeptidase C was purified from porcine skeletal muscle. 2. The mol. wt of the enzyme was found to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. 3. The optimum pH for the hydrolysis of L-leucine p-nitroanilide was around 7.0. 4. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. 5. The enzyme acted on the beta-naphthylamide derivatives of amino acids and oligopeptides.     

Purification and characterization of acylphosphatase erythrocyte isoenzyme from turkey muscle

An acylphosphatase has been purified from turkey muscle in a rapid and high-yield way. The enzyme has been characterized for structural, kinetic, and immunological parameters, as well as with regard to its stability to thermal, urea, and phenylglyoxal inactivation. The enzyme is quite different from the turkey muscular isoenzyme, and shows structural and kinetic properties that are very similar to those previously reported for the erythrocyte isoenzyme from human erythrocytes and from chicken muscle. From the data reported it appears that this enzyme corresponds to the acylphosphatase erythrocyte isoenzyme. Unlike the erythrocyte isoenzymes studied so far, this enzyme is able to cross-react with antibodies that are raised against the muscular isoenzyme.