Aminopeptidase P from human leukocytes.
Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four-step procedure. Buffy coats were the starting material. A M(r) of 140,000 was obtained by size-exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with M(r) of 71,000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N-terminal residues from peptides with N-terminal Xaa-Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di-, tri- and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg-Pro, Phe-Pro > Trp-Pro > Pro-Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C-terminal fragment (residues 201-206) of C-reactive protein, oxytocin fragment Tyr-Pro-Leu-Gly, morphiceptin, peptide Gly-Pro-Arg-Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin-6, was also demonstrated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10-phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy-phenylalanine, as well as several N-carbobenzoxy-proline-containing peptides, caused partial inhibition. The observed resistance of Gly-Pro, Pro-Gly, Pro-Phe and Pro-Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline-specific dipeptidases in the aminopeptidase-P preparation.
Rusu
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I
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Yaron
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A
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015850:Interleukin-6 / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D013379:Substrate Specificity
D015850:Interleukin-6; D008670:Metals; D010455:Peptides; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase