Thiol-dependent aminopeptidase (350,000 mol wt) as a marker of epidermal contamination in sweat.
L-Leucine 2-naphthylamide (Leu-NA) hydrolytic activity is increased 20-fold in eccrine sweat collected by simple scraping (SS) compared with sweat collected over the white petrolatum (Vaseline) barrier (clean sweat, CS) [Am. J. Physiol. 250 (Regulatory Integrative Comp. Physiol. 19): R691-R698, 1986]. Sephadex G-200 chromatography of SS but not that of CS showed a single peak of Leu-NA hydrolytic activity (at pH 8) at 350,000 mol wt. An enzyme with similar molecular weight was eluted from tape-stripped stratum corneum and from stripped skin in situ. Anion-exchange FPLC of the 350,000 fractions yielded a single Leu-NA hydrolase peak at pH 8 (pool IV), which also showed hydrolytic activity for benzoyl-L-arginine-2-naphthylamide (BANA). Both Leu-NA and BANA hydrolytic activities of pool IV were thiol dependent, inhibited by heavy metals, and activated by ethylenediaminetetraacetic acid. The pool IV enzyme also hydrolyzed L-lysine- and L-arginine-2-naphthylamide. The most prominent BANA hydrolase activity was seen in both SS and CS at pH 5.0 at 33,000, which was not associated with Leu-NA hydrolytic activity. Diethylaminoethyl cellulose chromatography of the 33,000 fractions yielded three peaks of BANA hydrolytic activity in SS but only one in CS, suggesting that this thiol-dependent BANA hydrolyzing enzyme in CS may be of sweat gland origin. We conclude that the 350,000 thiol-dependent Leu-NA-hydrolyzing aminopeptidase is one of the most prominent epidermal contaminants and thus is a useful marker of epidermal contamination in sweat samples.
D000328:Adult; D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D000078404:Epidermal Cells; D004817:Epidermis / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013542:Sweat / Q000166:cytology*
D002413:Cations, Divalent; D011480:Protease Inhibitors; D004492:Edetic Acid; D000626:Aminopeptidases; D004229:Dithiothreitol