Glycine β-naphthylamide

Crude lysosomal preparations from a cultured human skin fibroblast line were found to contain significant levels of a neutral pH hydrolase activity towards glycine--phenylalanine--beta-naphthylamide (NA), a substrate normally used for the assay of lysosomal dipeptidyl aminopeptidase I. However, the activity was chloride ion insensitive, nonlatent, and inhibitable by cationic detergents and amino acids. Assays of substrate selectivity, relative substrate affinity, pH and anion and cation sensitivity indicated the activity to be distinct from dipeptidyl aminopeptidases I (chloride-dependent hydrolysis of Pro-Phe-, Gly-Phe-, Gly-Arg-, and Pro-Arg-NA's at acid pH), II (Lys-Ala-NA hydrolysis), III (Arg-Arg-NA hydrolysis), and IV (Gly-Pro-NA hydrolysis). The lysosomal preparations also contained significant activity towards several amino acid--naphthylamides, notably Arg-NA. Only dipepidyl aminopeptidase I activity showed sensitivity to chloride anions, both dipeptidyl aminopeptidases I and II showed substantial latency, and none of the activities displayed a significant metal cation dependent.

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min(-1) for glutamate. At pH 7.5 (pH optimum) and 25 degrees C, the kinetic parameters for alanine para-nitroanilide were found to be k(cat) = 7600 min(-1) and K(m) = 14 mm. For alanine beta-naphthylamide, they were k(cat) = 860 min(-1) and K(m) = 6.7 mm. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the full-length gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.