title: Purification, substrate specificity, and classification of tripeptidyl peptidase II.
abstract: An extralysosomal tripeptide-releasing aminopeptidase was recently discovered in rat liver (Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. (1983) J. Biol. Chem. 258, 11622-11628). In the present work this tripeptidyl peptidase is shown to occur in several rat tissues and in human erythrocytes. The erythrocyte enzyme was purified about 80,000-fold from a hemolysate while the rat liver enzyme was purified about 4,000-fold from a homogenate. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions more than 90% of the protein was represented by a polypeptide of Mr 135,000 in both cases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed similar specific activity, and had a pH optimum around 7.5. A tryptic pentadecapeptide from the alpha-chain of human hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, was found to be sequentially cleaved by the erythrocyte enzyme into five tripeptides, beginning from the NH2 terminus. Chromogenic tripeptidylamides showed various rates of hydrolysis at pH 7.5. With Ala-Ala-Phe-4-methyl-7-coumarylamide, Km was 16 microM and Vmax 13 mumol min-1 . mg-1, comparable to the standard substrate Arg-Arg-Ala-Ser(32P)-Val-Ala values (Km 13 microM and Vmax 24 mumol . min-1 . mg-1). The tripeptidyl peptidase of human erythrocytes was classified as a serine peptidase from its irreversible inhibition by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala (Ki 1.5 microM). [3H]Diisopropylphosphate was incorporated to the extent of 0.7-0.9 mol/mol of Mr 135,000 subunit, which confirms the high purity of the enzyme.
mesh_terms: D000626:Aminopeptidases; D000818:Animals; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors; D051381:Rats; D011915:Rats, Inbred BUF; D012697:Serine Endopeptidases; D013379:Substrate Specificity
chemical_list: D002863:Chromogenic Compounds; D006454:Hemoglobins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D010450:Endopeptidases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases