Antigenic determinants of acylphosphatase from porcine skeletal muscle.

This study delves into the antigenic properties of acylphosphatase from pig skeletal muscle, identifying two antigenic determinants and exploring the cross-reactivities of acylphosphatases in horse and rabbit skeletal muscles. The research includes a comparative analysis of these enzymes’ molecular weight, charge, conformation, amino acid composition, and peptide sequences.

Examination of Antigenic Determinants

• The research initiated with an analysis of the precipitin reaction of the pig skeletal muscle’s acylphosphatase with rabbit antiserum. It established the existence of at least two antigenic determinants on the enzyme from pig’s muscle.

Investigation of Cross-reactivities

• The study then moved forward to examine the cross-reactivities of acylphosphatases from horse and rabbit skeletal muscles.
• During the double immunodiffusion with the antiserum, a full fusion was observed in the precipitin lines of the pig and horse enzymes. In contrast, the rabbit enzyme did not produce a precipitin line.
• When the pig enzyme was labeled and reacted with its antibody, both the pig and horse enzymes inhibited the reaction equally. Contrarily, the rabbit enzyme did not exhibit this inhibitory behavior.

Comparative Analysis of Enzymes

• A polyacrylamide gel electrophoresis assessed the molecular weight and net charge of the three enzymes, showing similarities among them.
• Further, the circular dichroism spectra of the enzymes reflected no conformational difference.
• However, discrepancies surfaced when the amino acid composition was analyzed. The rabbit enzyme demonstrated a different content of Glu, Gly, Lys, and Arg compared to the pig and horse enzymes.

Comparative Peptide Sequencing

• The enzyme differences were further explored by comparing the peptide maps of the two enzymes’ tryptic peptides. Four peptides of the rabbit enzyme were positioned differently compared to the pig enzyme.
• Amino acid analysis led to the sequencing of three out of the four peptides of both enzymes. Further examination of rabbit enzyme allowed to tentatively align its tryptic peptides based on the amino acid compositions and sequence homologies in comparison to the pig enzyme’s peptides.
• Out of five amino acid residues of the pig enzyme that were substituted in the rabbit enzyme, Arg-4 and Asp-28 were pinpointed as being part of the antigenic determinants.