Open AccessPurification and characterization of two Cl − ‐activated aminopeptidases hydrolysing basic termini from human skeletal muscle
Author(s) -
MANTLE David,
LAUFFART Brenda,
McDERMOTT John R.,
KIDD Alison M.,
PENNINGTON Ronald J. T.
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb08751.x
Subject(s) - chemistry , hydrolysis , aminopeptidase , size exclusion chromatography , chromatography , dipeptide , enzyme , biochemistry , divalent , gel electrophoresis , amino acid , stereochemistry , leucine , organic chemistry
Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion‐exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl − or Br − anions resulted in a 20‐fold or 10‐fold increasae in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05–5 mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 μM), p ‐hydroxymercuriphenylsulphonic acid (0.1 μM), EDTA (10 mM), 1,10‐phenanthroline (100 μM), N ‐ethylmaleimide (1 mM) and Bu t ‐Thr‐Phe‐Pro (0.5 mM). The molecular mass was 72000 Da (gel filtration). Only the arginyl and lysyl 7‐amino‐4‐methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate K m values for the reaction of I and II with these substrates (10–250 μM) were estimated as follow: Arg‐Ame, K 1 m = 70 μM; K II m = 270 μM; Lys‐Amc K I m = 280 μM, K II m = 400 μM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH 2 ‐terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH 2 ‐terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl − ‐requiring, thiol‐type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.