Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2(+)-dependent substrate specificity as a novel feature of the enzyme.
An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1,10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolysis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical Ki value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.
Yasuhara
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Yokosawa
H
H
Ishii
S
S
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D006868:Hydrolysis; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism; D012617:Sea Urchins; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity
D010455:Peptides; D000626:Aminopeptidases; D002118:Calcium