Isolation and properties of digestive carboxypeptidases from midguts of larvae of the black carpet beetle Attagenus megatoma

Abstract

Two cationic carboxypeptidase fractions (CMS-G-1 and CMS-G-2) were isolated from the midgut homogenate of larvae of the black carpet beetle, Attagenus megatoma, through a series of ion exchange and affinity gel chromatographic steps. Fraction CMS-G-2 was highly active against the substrates hippuryl- dl-β-phenyllactate, N- carbobenzoxy-glycyl- l-phenylalanine , and N- carbobenzoxy-glycyl- l-tyrosine and was thus similar in specificity to bovine carboxypeptidase A. Fraction CMS-G-1 was most active against N- carbobenzoxy- l-valyl- l-leucine and N- carbobenzoxy-glycyl- l-methionine . Fraction CMS-G-1 also hydrolysed (in decreasing order) blocked peptides with carboxyl terminal l-isoleucine, l-leucine, l-alanine, and l-valine but was only slightly active against the ester and peptide substrates of carboxypeptidase A. Neither CMS-G-1 nor CMS-G-2 hydrolysed N- carbobenzoxy-glycyl- l-proline or glycyl-glycine. Between CMS-G-1 and CMS-G-2, at least four bands with peptidase activity were resolved on a cationic polyacrylamide gel system. The terminal and penultimate amino acids of the peptides were equally important in determining the specificity of individual peptidase bands. Fractions CMS-G-1 and CMS-G-2 each had a single, separate activity band with the substrate N- carbobenzoxy-glycyl- l-leucine . These two peptidases had molecular weights of about 35,500 and were most active at pH 7.5 to 8.5. Both enzymes were inhibited by the metal chelators EDTA and 1,10-phenanthroline and also by heavy metals such as Cu 2+, Hg 2+ and Cd 2+. The two enzymes thus appear to be metalloproteins with active sulphydryl groups.