Enzymatic properties of human aminopeptidase A. Regulation of its enzymatic activity by calcium and angiotensin IV.
Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ up- or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca2+ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases. Taken together, these results suggest that there exists a complex regulation of the enzymatic activity of APA, which may contribute to homeostasis such as regulation of blood pressure, maintenance of memory, and normal pregnancy by controlling the concentrations of peptide substrates.
Goto
Yoshikuni
Y
Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama, 351-0198, Japan.
Hattori
Akira
A
Ishii
Yasuhiro
Y
Mizutani
Shigehiko
S
Tsujimoto
Masafumi
M
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000737:chemistry / Q000502:physiology; D016367:Baculoviridae / Q000235:genetics / Q000276:immunology; D002118:Calcium / Q000737:chemistry* / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D043384:Glutamyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity
D010455:Peptides; D011994:Recombinant Proteins; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D043384:Glutamyl Aminopeptidase; D002118:Calcium