Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M.
Aminopeptidase M (AmM; EC 3.4.11.2) is a membrane-bound peptidase present on renal brush border and vascular plasma membrane. In the present study, AmM, purified from rabbit kidney cortex, produced a single immunoprecipitin line against AmM antisera, hydrolyzed alanyl-, leucyl- and arginyl-beta-naphthylamides at rates of 5.1 +/- 0.5, 3.9 +/- 0.5 and 2.6 +/- 0.3 mumol/min/mg, respectively, exhibited little or no alpha-glutamyl-, aspartyl- or glycyl-prolyl-naphthylamidase activities (less than or equal to 0.14 mumol/min/mg), and was inhibited by o-phenanthroline, amastatin (IC50 = 400 nM) and bestatin (IC50 = 6 microM). The alanyl-naphthylamidase activity of unfractionated rabbit plasma was found to be identical to purified AmM regarding relative rates of hydrolysis of alanyl-, leucyl- and arginyl-naphthylamides (100:79:42), pH optimum, and inhibition profile. In comparative studies with the purified enzyme, immunoreactive AmM accounted for essentially all of the alanyl-2-naphthylamidase activity of rabbit plasma. N-Terminal metabolism of (Met5)enkephalin by purified renal AmM was 3.92 +/- 0.69 mumol/min/mg, followed by somatostatin (1.25 mumol/min/mg), hepta(5-11)substance P (1.14 +/- 0.13 mumol/min/mg), (Asn1)angiotensin II (1.11 +/- 0.06 mumol/min/mg), angiotensin III (0.45 +/- 0.04 mumol/min/mg) and des(Asp1)-angiotensin I (0.36 +/- 0.04 mumol/min/mg). In contrast, substance P, bradykinin, (Sar1,Ala8)angiotensin II and neurokinin analogs containing modified N-termini (e.g. Ac-Arg) were resistant to hydrolysis by AmM. Peptide degradation was optimal at neutral pH and was inhibited by amastatin (IC50 = 200 nM) and bestatin (IC50 = 5 microM). Apparent Km values ranged from 15.7 +/- 0.4 microM for angiotensin III to 102 +/- 2 microM for (Met5)enkephalin. These data support a significant role for vascular and plasma AmM in the metabolism of circulating vasoactive peptides.
Ward
P E
PE
Department of Physiology, Ohio State University, Columbus 43210.
Benter
I F
IF
Dick
L
L
Wilk
S
S
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018826:CD13 Antigens; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004723:Endorphins / Q000378:metabolism; D007123:Immunoelectrophoresis, Two-Dimensional; D007672:Kidney Cortex / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D014660:Vasoactive Intestinal Peptide / Q000097:blood / Q000378:metabolism*
D004723:Endorphins; D014660:Vasoactive Intestinal Peptide; D000626:Aminopeptidases; D018826:CD13 Antigens