Endooligopeptidase A activity in rabbit heart: generation of enkephalin from enkephalin containing peptides.
Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met5] enkephalin and [Leu5]enkephalin from small enkephalin containing peptides. They also hydrolyze the Phe5-Ser5 bond of bradykinin and the Arg8-Arg9 bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of Mr = 73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr = 72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulin reacts with a polypeptide corresponding to Mr = 50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A.
Cicilini
M A
MA
Department of Physiological Sciences, Universidade Federal do Espirito Santo, ES, Brazil.
Ribeiro
M J
MJ
de Oliveira
E B
EB
Mortara
R A
RA
de Camargo
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D004745:Enkephalins / Q000096:biosynthesis*; D007202:Indicators and Reagents; D008666:Metalloendopeptidases; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009479:Neuropeptides / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D004745:Enkephalins; D007202:Indicators and Reagents; D009479:Neuropeptides; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase