Identification of three continuous antigenic sites in horse muscle acylphosphatase

The main antibody-combining sites of horse skeletal muscle acylphosphatase were mapped by preparing and purifying CNBr, tryptic and peptic peptides from the pure enzyme, and looking for the immunoreactivity of each peptide by the dot-immunobinding assay using specific polyclonal antienzyme antibodies previously purified by immunoaffinity chromatography. The immunoreactive peptides were identified on the basis of either their elution times in the fingerprint analysis or amino acid composition, or both, by comparison with the known enzyme amino acid sequence. All the CNBr as well as two tryptic and two peptic peptides were immunopositive, leading to identification of three main continuous antigenic sites on the enzyme molecule. The strong inhibition (92%) of the antigen-antibody reaction carried out in the presence of antibodies previously incubated with the immunoreactive peptide mixture supports the possibility that, at our experimental condition, the three identified antigenic domains contain the main antigenic determinants of the enzyme. The relationship between structure and antigenicity of the immunoreactive peptides is discussed in detail.     

Properties of Cys21-mutated muscle acylphosphatases

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed inEscherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.     

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k_F^H2O = 72,600 s^− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.     

Proteome-Level Interplay between Folding and Aggregation Propensities of Proteins

With the advent of proteomics, there is an increasing need of tools for predicting the properties of large numbers of proteins by using the information provided by their amino acid sequences, even in the absence of the knowledge of their structures. One of the most important types of predictions concerns whether proteins will fold or aggregate. Here, we study the competition between these two processes by analyzing the relationship between the folding and aggregation propensity profiles for the human and Escherichia coli proteomes. These profiles are calculated, respectively, using the CamFold method, which we introduce in this work, and the Zyggregator method. Our results indicate that the kinetic behavior of proteins is, to a large extent, determined by the interplay between regions of low folding and high aggregation propensities.     

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.     

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.     

Rat muscle acylphosphatase: Purification, amino sequence, and immunological characterization

Acylphosphatase was purified from rat skeletal muscle essentially by gel filtration and high-performance ion-exchange chromatography. The complete amino acid sequence was reconstructed by using the sequence data obtained from tryptic, peptic, andS. aureus V8 protease peptides. The protein consists of 96 amino acid residues and is acetylated at the NH₂-terminus. The immunological cross-reactivity of acylphosphatase from rat and horse skeletal muscle was examined by ELISA. The reaction with rabbit antiserum revealed the presence of at least five antigenic sites on rat enzyme, two of which are common to horse muscle enzyme. Anti-rat antibodies also recognize the peptide that corresponds to the initial part of the molecule, which varies greatly from equine enzyme. Two completely new antigenic sites are herein described: the first can be considered the main antigenic site and is located within positions 21–36, the second is in the COOH-terminal part of the molecule. A mixture of immunoreactive peptides gives strong antibody-antigen reaction inhibition (94%).     

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.     

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.