Degradation of thymopentin by human lymphocytes: evidence for aminopeptidase activity.
Thymopentin (Arg-Lys-Asp-Val-Tyr) was shown to be degraded in vitro by human lymphocytes into two main fragments; the tetrapeptide Lys-Asp-Val-Tyr and the tripeptide Asp-Val-Tyr. Degradation products were identified by HPLC and amino-acid analysis. Analysis of the time-course of degradation revealed a 'stepwise' degradative event beginning at the N-terminal. The degradation of thymopentin after the first 10 min, as well as the formation of the tetrapeptide (5-30 min) were essentially curvilinear. Degradation of the tripeptide, was linear. Upon screening a panel of compounds that inhibit enzymatic activity, bestatin, amastatin and 1,10-phenanthroline were shown to be the most effective. Bestatin and amastatin caused an 85-90% inhibition of thymopentin degrading activity with IC50 values of 7.1 x 10(-6) M and 4.5 x 10(-9) M, respectively. 1,10-Phenanthroline completely inhibited the degradative process with an IC50 of 2 x 10(-4) M. When the tetrapeptide Lys-Asp-Val-Tyr was used as the starting substrate, similar IC50 values were seen for amastatin, bestatin and 1,10-phenanthroline. The importance of divalent metal ions in the degradative event was demonstrated not only by the effect of 1,10-phenanthroline, but also by the ability of Zn2+ and Co2+ to reverse the inhibition of 1,10-phenanthroline (at its IC50) to activities near control values (no inhibitor). These data strongly suggest that an aminopeptidase(s) is responsible for the degradative activity.
Amoscato
A A
AA
Department of Surgery, University of Cincinnati College of Medicine, OH 45267-0558.
Balasubramaniam
A
A
Alexander
J W
JW
Babcock
G F
GF
D000626:Aminopeptidases / Q000097:blood*; D000900:Anti-Bacterial Agents; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000097:blood*; D010455:Peptides; D010618:Phenanthrolines / Q000494:pharmacology; D016305:Thymopentin; D013946:Thymopoietins / Q000097:blood*; D013951:Thymus Hormones / Q000097:blood*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D010618:Phenanthrolines; D013946:Thymopoietins; D013951:Thymus Hormones; C016977:amastatin; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex; D016305:Thymopentin; C025205:1,10-phenanthroline