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.     

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.     

N-acetylserine in horse muscle acylphosphatase.

A ninhydrin-negative peptide fraction obtained from tryptic digest of carboxymethyl acylphosphatase was isolated by chromatography on a column of PA 28 Beckman resin and analysed for the amino acid composition. Degradation with carboxypeptidase B and A indicated that the sequence of this peptide was: X-Thr-Ala-Arg. The amino-terminal residue was identified as N-acetylserine by high voltage electrophoresis. It is therefore suggested that the sequence of the NH2-terminal portion of CM-acylphosphatase is N-acetyl-Ser-Thr-Ala-Arg. Digestion with carboxypeptidase A and B indicated also that the COOH-terminal portion of CM-acylphosphatase is-Arg-Tyr-OH.     

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.     

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.     

Identification of mutations at the antigenic and glycosylation sites in hemagglutinin protein of H5N1 strain

Hemagglutinin (HA) is the principal antigen, present on the viral surface. It is the primary target for neutralizing antibodies. In this paper, wehave carried out studies on human hemagglutinin protein from H5N1 strain with homologous hemagglutinin from non-human sources of H5N1strains. In all strains, part of the antigenic site (128-141) predicted by computer program “Antigenic”, corresponds to immunodominant site Sa ofH1 subtype. In {type:entrez-protein,attrs:{text:AAF02304,term_id:6048753,term_text:AAF02304}}AAF02304 strain, A156→S156 mutation lies at the antigenic subsite of site 2 that corresponds to site B in the H3 subtype. Insome strains of non-human origins, there are mutations at the antigenic sites. Interestingly, in {type:entrez-protein,attrs:{text:AAY56367,term_id:66775625,term_text:AAY56367}}AAY56367 strain mutation L138→H138 lies at thereceptor binding site, which also overlaps the antigenic site. Therefore, this amino acid substitution may influence both the specificity of receptorrecognition and antibody binding. Seven potential glycosylation sites in human HA and in some strains of non-human sources have beenpredicted by computer program, Scan Prosite. In some strains of HA from non-human sources because of mutation, an additional glycosylationsite appeared at the antigenic site. Therefore in these strains the oligosaccharides will mask the surface of HA as well as antigenic site. Hencethese strains will not be recognized by host immune system.     

Purification of horse muscle acylphosphatase antibodies by affinity chromatography

Horse muscle acylphosphatase antibodies were obtained by immunizing rabbits with the highly purified antigen cross-linked with glutaraldehyde. Specific antibodies were purified from the immunoglobulin fraction by affinity chromatography using a matrix coupled with the pure antigen as immunoadsorbent. The purified antibodies were partially characterized by immunodiffusion and immunoprecipitin techniques. These antibodies could be used to study aspects of the muscle acylphosphatase structure, localization and other biological properties.     

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.     

Alterations in antigenic structure of gonadotropins following deglycosylation

Radioimmunological techniques were utilized to probe possible changes in conformation of gonadotropins (human chorionic gonadotropin-hCG; and ovine luteinizing hormone—oLH) following chemical deglycosylation (DG-hCG and DG-LH). All antisera produced in rabbits, rats or mice contained antibodies that were specific to the deglycosylated hormones with the native hormones showing weak and non-parallel cross-reaction (<5%), but with rabbit antibodies to native hormones the deglycosylated hormones were fully reactive. Using hCG, asialo-hCG (A-hCG) and DG-hCG, we have shown that removal of sugars internal to sialic acid is required to produce these specific antibodies. These are in complete agreement with the observations that extensive deglycosylation of these hormones is necessary to induce changes in biological activity at the cellular level. Based on these data, we suggest that chemical deglycosylation results in changes in antigenic structure of these hormones by generation of new determinants or exposure of previously buried sites and these changes are of no consequence to receptor recognition.     

Stability and kinetic behavior of carboxymethylated horse muscle acylphosphatase.

Horse muscle acylphosphatase consists of a main chain S-S bound to glutathione. It was found that removal of the glutathione by reduction and successive carboxymethylation of the only cysteine of the main chain affects the stability of the enzyme, mainly with respect to thermal inactivation. On the other hand, the kinetic properties of the enzyme are affected very little.     

Inhibition of horse muscle acylphosphatase by pyridoxal 5'-phosphate.

It has been shown that horse muscle acylphosphatase is inhibited by pyridoxal 5'-phosphate and that the inhibition is pH dependent, reversible and competitive with respect to substrate binding. Spectral analysis on the EI complex demonstrates the presence of a Schiff base. Reduction of the pyridoxal 5'-phosphate-inhibited enzyme with sodium borohydride, followed by amino acid analysis, produces a diminution of the free lysine peak and the appearance of a new peak corresponding to epsilon-pyridoxyllysine. The results suggest that there is at least one NH2-lysyl residue of horse muscle acylphosphatase at or near the active site of the enzyme.     

Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy

It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to locate the two segments within the primary structure (residues 22 to 35 and 55 to 66), thus providing the sequential assignment. The assignment of the side-chains was completed and a list of all nuclear magnetic resonance constraints within the two segments (126 intra- and 180 interresidue distances, 21 torsion angles phi and 19 hydrogen bonds) was produced. Distance geometry calculation shows that each segment forms an alpha-helix. The mutual orientation of the two helices was established subsequently.     

Comparison of the antigenic peptides between human prostatic and lysosomal acid phosphatases

The relative antigenicity (capacity to bind antibodies raised against the intact prostatic acid phosphatase) of the selected peptides from human prostatic and lysosomal acid phosphatases was evaluated in a competitive assay. Both prostatic and lysosomal acid phosphatases were shown to possess similar antigenic determinants on both terminal regions, along with more similarity on NH₂-terminal peptide than COOH-terminal site. At least one additional antigenic site is present at the internal region of prostatic acid phosphatase, since the mixture of both amino- and carboxyl-terminal peptides exhibited only 70% inhibition.     

Stability of horse muscle acylphosphatase to heat and to urea.

The thermal stability of horse muscle acylphosphatase was investigated by measuring the inactivation constants at various pH and temperature values, and by differential spectra technique. This enzyme has high thermal stability in an acidic environment but is inactivated in an alkaline medium. It was found that the enzyme can be protected against such inactivation at pH 8.0 by increasing its concentration and the ionic strength of the solution. The effect of high urea concentrations on stability was also measured. It was found that spectral changes at 230 nm are related to urea inactivation of the enzyme, and that the enzymatic activity can be instantly and almost completely restored by dilution of the urea.     

Computational binding assays of antigenic peptides

Summary Computer models can be combined with laboratory experiments for the efficient determination of (i) peptides that bind MHC molecules and (ii) T-cell epitopes. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures. This requires the definition of standards and experimental protocols for model application. We describe the requirements for validation and assessment of computer models. The utility of combining accurate predictions with a limited number of laboratory experiments is illustrated by practical examples. These include the identification of T-cell epitopes from IDDM-, melanoma-and malaria-related antigens by combining computational and conventional laboratory assays. The success rate in determining antigenic peptides, each in the context of a specific HLA molecule, ranged from 27 to 71%, while the natural prevalence of MHC-binding peptides is 0.1–5%.     

Computational binding assays of antigenic peptides

Computer models can be combined with laboratory experiments for the efficient determination of (i) peptides that bind MHC molecules and (ii) T-cell epitopes. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures. This requires the definition of standards and experimental protocols for model application. We describe the requirements for validation and assessment of computer models. The utility of combining accurate predictions with a limited number of laboratory experiments is illustrated by practical examples. These include the identification of T-cell epitopes from IDDM-, melanoma- and malaria-related antigens by combining computational and conventional laboratory assays. The success rate in determining antigenic peptides, each in the context of a specific HLA molecule, ranged from 27 to 71%, while the natural prevalence of MHC-binding peptides is 0.1–5%.     

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.     

Interaction between acylphosphatase and SERCA in SH-SY5Y cells

Ca²⁺ transport by sarco/endoplasmic reticulum, tightly coupled with the enzymatic activity of Ca²⁺-dependent ATPase, controls the cell cycle through the regulation of genes operating in the critical G₁ to S checkpoint. Experimental studies demonstrated that acylphosphatase actively hydrolyses the phosphorylated intermediate of sarco/endoplasmic reticulum calcium ATPase (SERCA) and therefore enhances the activity of Ca²⁺ pump. In this study we found that SH-SY5Y neuroblastoma cell division was blocked by entry into a quiescent G₀-like state by thapsigargin, a high specific SERCA inhibitor, highlighting the regulatory role of SERCA in cell cycle progression. Addition of physiological amounts of acylphosphatase to SY5Y membranes resulted in a significant increase in the rate of ATP hydrolysis of SERCA. In synchronized cells a concomitant variation of the level of acylphosphatase isoenzymes opposite to that of intracellular free calcium during the G₁ and S phases occurs. Particularly, during G₁ phase progression the isoenzymes content declined steadily and hit the lowest level after 6 h from G₀ to G₁ transition with a concomitant significant increase of calcium levels. No changes in free calcium and acylphosphatase levels upon thapsigargin inhibition were observed. Moreover, a specific binding between acylphosphatase and SERCA was demonstrated. No significant change in SERCA-2 expression was found. These findings suggest that the hydrolytic activity of acylphosphatase increase the turnover of the phosphoenzyme intermediate with the consequences of an enhanced efficiency of calcium transport across endoplasmic reticulum and a subsequent decrease in cytoplasmic calcium levels. A hypothesis about the modulation of SERCA activity by acylphosphatase during cell cycle in SY5Y cells in discussed.     

Predicted secondary structure of horse muscle acylphosphatase. Comparison with circular dichroism measurements

We have predicted the secondary structure of horse muscle acylphosphatase by the statistical method of Chou and Fasman. In addition, we have studied the circular dichroism spectra of the enzyme, obtaining values for comparison to the predicted results. Discrepancies were found for the alpha-helix content estimated by the two methods.