Surface aminopeptidase activity of human lymphocytes. I. Biochemical and biologic properties of intact cells.
Surface aminopeptidase activity in intact lymphocytes was studied and was shown to have the following properties when alanine-p-nitroanilide was used as substrate: 1) The activity was surface associated and not secreted as determined by extracellular location of product and the effect of proteases and diazotized sulfanilic acid on enzyme activity. 2) The enzyme activity was shown to have a pH optimum of 7.4 to 8.0. 3) Enzyme activity was shown to be inhibited by amastatin, bestatin, and 1,10 phenanthroline. Inhibition by amastatin consisted of a high-affinity component (Ki = 3.5 x 10(-6) M) which accounted for approximately 20% of the total activity and a low-affinity component (Ki = 3.5 x 10(-5) M) which accounted for the remainder suggesting that two forms of aminopeptidase exist. Only a single component of inhibition was seen with bestatin (Ki = 3.5 x 10(-6) M) and 1,10 phenanthroline (Ki = 2.0 x 10(-4) M) which accounted for 80 and 90% of the total enzyme activity, respectively. Unlike the competitive inhibitors bestatin and amastatin, inhibition by 1,10 phenanthroline was shown to be non-competitive. Finally, surface aminopeptidase activity essentially doubled in the presence of PHA (10 micrograms/ml) or Con A (10 micrograms/ml), at 72 h. This enhancing effect was shown to be dose dependent, time dependent, and mitogen dependent and correlated with the cellular state of activation as determined by [3H]TdR incorporation.
Amoscato
A A
AA
Department of Surgery, University of Cincinnati College of Medicine, OH 45267-0558.
Alexander
J W
JW
Babcock
G F
GF
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000900:Anti-Bacterial Agents; D002413:Cations, Divalent; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides; D010618:Phenanthrolines / Q000494:pharmacology; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D002413:Cations, Divalent; D009842:Oligopeptides; D010455:Peptides; D010618:Phenanthrolines; C016977:amastatin; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex