Bovine testis acylphosphatase: Purification and amino acid sequence

Two acylphosphatase molecular forms have been isolated from bovine testis. Their amino acid sequence was determined. One (ACY1) consists of 98 amino acid residues, while the other one (ACY2) consists of 100 amino acid residues. Both molecular forms are N-acetylated and differ only in the amino terminus. ACY2 has an additional Ser-Met tail with respect to ACY1. Both ACY1 and ACY2 are organ-common type isoenzymes and thus differ for about half of the amino acid positions from the previously sequenced bovine muscle isoenzyme.     

The primary structure of chicken muscle acylphosphatase isozyme Ch1

The amino acid sequence of chicken muscle acylphosphatase isozyme Ch1 was determined. The protein consists of 102 amino acid residues, does not contain histidine, and the NH2-terminus is acetylated: Ac-Ser-Ala-Leu-Thr-Lys-Ala-Ser-Gly-Ser- Leu-Lys-Ser-Val-Asp-Tyr-Glu-Val-Phe-Gly-Arg-Val-Gln-Gly-Val-Cys-Phe-Arg- Met- Tyr-Thr-Glu-Glu-Glu-Ala-Arg-Lys-Leu-Gly-Val-Val-Gly-Trp-Val-Lys-Asn- Thr- Ser-Gln-Gly-Thr-Val-Thr-Gly-Gln-Val-Gln-Gly-Pro-Glu-Asp-Lys-Val-Asn-Ala- Met- Lys-Ser-Trp-Leu-Ser-Lys-Val-Gly-Ser-Pro-Ser-Ser-Arg-Ile-Asp-Arg-Thr-Lys- Phe-Ser- Asn-Glu-Lys-Glu-Ile-Ser-Lys-Leu-Asp-Phe-Ser-Gly-Phe-Ser-Thr-Arg-Tyr-OH. This sequence differs in 44% of the total positions from the other isozyme (Ch2) of chicken muscle acylphosphatase (Ohba et al., the accompanying paper). The sequence of Ch1 has three substitutions from that of turkey muscle acylphosphatase; these are Ser from Ala at position 9, Ser from Arg at 47, and Lys from Asn at 83. The sequence has about 80% homology with those mammalian muscle acylphosphatases.     

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.     

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.     

A new acylphosphatase isoenzyme from human erythrocytes: purification, characterization, and primary structure

A new acylphosphatase from human erythrocytes was isolated by an original purification procedure. It is an isoenzyme of the well-characterized human skeletal muscle acylphosphatase. The erythrocyte enzyme shows hydrolytic activity on acyl phosphates with higher affinity than the muscle enzyme for some substrates and phosphorylated inhibitors. The sequence was determined by characterizing the peptides purified from tryptic, peptic, and Staphylococcus aureus V8 protease digests of the protein, and it was found to differ in 44% of the total positions as compared to the human muscle enzyme. About one-third of these differences are in the form of strictly conservative replacements. The protein consists of 98 amino acid residues; it has an acetylated NH2-terminus and does not contain cysteine: (sequence in text).     

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.     

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.     

Effect of exogenously added acylphosphatases on inositol lipid metabolism in human platelets

In this paper we demonstrate that human platelets contain an acylphosphatase isoenzyme. We then investigated the effect of exogenously added human muscle and erythrocyte acylphosphatases on inositol lipid content in human platelets permeabilized with saponin. Alterations in the level of the polyphosphoinositides were observed: in particular, the levels of phosphatidylinositol 4,5-bisphosphate, and of phosphatidylinositol 4-monophosphate were decreased, whereas the level of phosphatidylinositol was increased. These results suggest that acylphosphatases promote polyphosphoinositide dephosphorylation, possibly through intracellular Ca2+ mobilization.     

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).     

The primary structure of chicken muscle acylphosphatase isozyme Ch2

The amino acid sequence of one, Ch2, of the two isozymes of chicken muscle acylphosphatase was determined. It consists of 98 amino acid residues with N-acetylalanine at the amino(N)-terminus and contains no cysteine: Ac-Ala-Gly-Ser-Glu- Gly-Leu-Met-Ser-Val-Asp-Tyr-Glu-Val-Ser-Gly-Arg-Val-Gln-Gly-Val-Phe-Phe- Arg- Lys-Tyr-Thr-Gln-Ser-Glu-Ala-Lys-Arg-Leu-Gly-Leu-Val-Gly-Trp-Val-Arg-Asn- Thr- Ser-His-Gly-Thr-Val-Gln-Gly-Gln-Ala-Gln-Gly-Pro-Ala-Ala-Arg-Val-Arg-Glu- Leu- Gln-Glu-Trp-Leu-Arg-Lys-Ile-Gly-Ser-Pro-Gln-Ser-Arg-Ile-Ser-Arg-Ala-Glu- Phe- Thr-Asn-Glu-Lys-Glu-Ile-Ala-Ala-Leu-Glu-His-Thr-Asp-Phe-Gln-Ile-Arg-Lys- COOH. The sequence differs in 44% of the total positions from the other isozyme, Ch1. Comparison of the sequence and the predicted conformational profile of Ch2 with those of Ch1 suggests that they share a common evolutionary origin and appear to have retained similar conformations throughout their evolutionary development.     

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.     

The complete amino acid sequence of horse muscle acylphosphatase

The amino acid sequence of horse muscle acylphosphatase is given in the present paper. The carboxymethylated enzyme consists of a single polypeptide chain of 98 amino acid residues with an acetyl group blocking the NH2 terminus and a tyrosine at the COOH terminus. The calculated molecular weight of the native protein, a mixed disulfide with glutathione, is 11,365. The carboxymethylated protein was cleaved by cyanogen bromide. The three expected fragments were purified; moreover, an additional fragment, derived from a partial failure of cleavage at methionine-24, was purified and characterized. The structures of the cyanogen bromide fragments were established by subfragmentation with endopeptidases, and the sequences of the overlapping subfragments were determined. From the results, it was possible to order the peptides within the sequence and then to establish the complete primary structure of the enzyme.     

Guinea pig acylphosphatase: The amino acid sequence

We determined the primary structure of guinea pig skeletal muscle acylphosphatase, using the high degree of homology with several vertebrate acylphosphatases to obtain correct alignment of the complete series of tryptic peptides. Their sequences were obtained mainly by Edman degradation; FAB mass spectrometry was used to identify the acyl group blocking the NH₂-terminal residue and to elucidate the structure of the NH₂-terminal tryptic peptide. The comparison among acylphosphatase sequences from skeletal muscle of several vertebrate species is presented and discussed.     

Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.     

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.     

Complete amino acid sequence of human transforming growth factor type beta 2

The complete amino acid sequence of human type beta 2 transforming growth factor (hTGF-beta 2) was determined by automated Edman degradation of S-pyridylethylated hTGF-beta 2 and selected fragments. Cleavage of hTGF-beta 2 by enzymatic and chemical techniques established all the fragments in an unambiguous sequence. Human TGF-beta 2 consists of two disulfide-linked, identical subunits. Each hTGF-beta 2 subunit is a single-chain polypeptide of 112 residues, with a calculated molecular weight of 12,720. Human TGF-beta 2 displays 71.4% sequence homology with the functionally related human TGF-beta 1, and is distantly related (23-40% amino acid identity) to porcine inhibins and activins, the carboxyl-terminal regions of human Müllerian inhibiting substance, and the putative decapentaplegic gene complex protein of Drosophila.