Enzymatic Properties of Human Aminopeptidase A

Abstract

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 Ca 2؉ , the preferential release of acidic amino acid residues was observed in the presence of Ca 2؉ . Moreover, Ca 2؉ 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 Ca 2؉ . Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca 2؉ . These results suggest that Ca 2؉ 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 Ca 2؉ 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.

Laeverin/Aminopeptidase Q, a Novel Bestatin-sensitive Leucine Aminopeptidase Belonging to the M1 Family of Aminopeptidases

“…Although leupeptin (a serine protease inhibitor) and pepstatin (an asparatic protease inhibitor) suppressed sLaeverin/APQ activity significantly only at high concentration (1 mM), E64 (a cysteine protease inhibitor) and p-(4-amidinophenyl)methanesulfonyl fluoride (a serine protease inhibitor) had only small inhibitory effects at 1 mM. Taken together, these data revealed that Laeverin/APQ is indeed a novel M1 aminopeptidase having enzymatic properties characteristic to the M1 family (7,13,23,24).…”

“…inhibitory effects on the enzymatic activity of sLaeverin/APQ, which is consistent with the other M1 aminopeptidases so far tested. In our previous work, Ca 2ϩ was shown to modulate the enzymatic activity of APA (24). However, no significant effect of Ca 2ϩ on the enzymatic activity of sLaeverin/APQ was observed up to 3 mM.…”

“…4C), suggesting that Ang IV was not a substrate of the enzyme. Ang IV was shown to inhibit APA (24). We therefore tested whether or not Ang IV is an inhibitor of Laeverin/APQ and found that it is a competitive inhibitor of Laeverin/APQ with a K i value of 8.96 Ϯ 1.07 M (data not shown).…”

“…Based on our experience (24), peptides that show Ͼ50% inhibition of the hydrolysis of the fluorogenic substrate at 100 M in this assay could be assumed to be substrates of the enzyme. Therefore, we examined the cleavage of candidate peptides by detecting degraded peptides after separating on reverse-phase HPLC.…”

Secretion of Endoplasmic Reticulum Aminopeptidase 1 Is Involved in the Activation of Macrophages Induced by Lipopolysaccharide and Interferon-γ

“…In addition, Q181D ERAP1, which showed different substrate specificity from the wild-type enzyme and cleaved basic amino acids preferentially, also had only a marginal effect (16); thus, the enzymatic activity of wild-type ERAP1 is required for the enhancement of phagocytosis. We then examined the effects of other M1 aminopeptidases, such as placental leucine aminopeptidase (P-LAP)/insulin-regulated aminopeptidase/oxytocinase, laeverin/aminopeptidase Q (APQ), and aminopeptidase A (APA), on the uptake of IgG-coated latex beads (17)(18)(19). Because these three enzymes are membrane-bound proteins, we prepared recombinant soluble forms for this purpose.…”