Purification and Characterization of Human Placental
Aminopeptidase A

Yamada, Reiko; Mizutani, Shigehiko; Kurauchi, O.; Okano, Kanemichi; Imaizumi, Hiromichi; Narita, O; Tsutsumi, Yutaka

doi:10.1159/000469167

Cited by 40 publications

(25 citation statements)

References 9 publications

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“…In contrast, Zn2+ inhibited the enzymatic activity on both cell types. The data is in agreement with previous reports that enzymatic activity of purified APA is increased by alkaline earth metals such as Ca2+, Sr2i, and Ba2+ but decreased by transition metals such as Hg2+, Cu2+, Ni2+, Zn2+, and Cd2+ (19,20). Interestingly, when cells were preincubated with PBS containing 50 mM EDTA to remove Zn2+ and other metal ions from the APA molecules, a low concentration of Zn2+ (0.2 mM) increased enzymatic activity on both cell types in the presence or absence of 1 mM CaCl2 (Fig.…”

Section: Methodssupporting

confidence: 93%

Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

Wang

Cooper

1993

Proc. Natl. Acad. Sci. U.S.A.

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The murine BP-1 antigen (also called 6C3) is a homodimeric, phosphorylated cell surface glycoprotein that is expressed on immature B-lineage cells, bone marrow stromal cell lines, thymic cortical epithelial cells, endothelial cells, enterocytes, and renal proximal tubular cells. The amino acid sequence deduced from a BP-1 cDNA predicted a type II integral membrane protein with a zinc-binding motif (His-GluXaa-Xaa-His) found in zinc-dependent metallopeptidases, and functional analysis suggested that BP-1 is aminopeptidase A [APA; L-a-aspartyl(L-ar-glutamyl)-peptide hydrolase, EC 3.4.11.71. Here we constructed an expression vector in which the BP-1 cDNA was placed downstream from the SRa promoter and used this construct to transfect COS-7 and Ltkcells. Both transfectants expressed the BP-1 antigen on the cell surface and APA activity. The enzymatic activity of recombinant APA was increased by Ca2+ and inhibited by Zn2+, consistent with previous reports with purified APA. Point mutation of one of the histidine residues in the zinc-binding motif to phenylalanine completely abolished APA enzymatic activity, suggesting the structure of the zinc-binding motif of APA is critical for catalytic activity. Both wild-type and mutant BP-1 were glycosylated, transported to the cell surface, and possessed molecular weights similar to native BP-1 molecules on the murine 18.81 pre-B-cell line. The successful expression of both wild-type and mutant APA should allow more precise analysis of the diverse physiological roles of this ectoenzyme.The differentiation of B cells requires interaction of lymphocyte precursors with other cell types in the microenvironments of fetal liver and adult bone marrow. Cell surface proteins expressed by developing B-lymphocyte precursors are thought to be involved in this highly complex, regulated process. The BP-1 antigen was identified as a homodimeric, phosphorylated cell surface glycoprotein on pre-B-cells and newly formed B cells in mouse bone marrow (1-3). This molecule, independently identified as a pre-B-cell neoplasiaassociated antigen and named 6C3 in another laboratory (4), is also expressed on certain bone marrow stromal cell lines and a subpopulation of thymic cortical epithelial cells (5-7).In mice, expression of the BP-1 (6C3) antigen on bone marrow-derived stromal cell lines correlates with their ability to support pre-B-cell growth (5). Interleukin 7 (IL-7) preferentially induces proliferation of bone marrow-derived pre-Bcells and up-regulates their expression of the BP-1 antigen (7,8), whereas its expression is extinguished in mature B lymphocytes and plasma cells (1). All of these observations suggest that the BP-1 molecule may play a crucial role in regulating growth and differentiation of early B-lineage cells.The BP-1 cDNA has been cloned recently (9), and the deduced amino acid sequence predicts a type II integral membrane protein with a zinc-binding motif-uncharged residue (Xaa)-Xaa-His-Glu-Xaa-Xaa-His-Xaa-hydrophobic residue-that is conserved among members of the zincd...

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Section: Methodssupporting

confidence: 93%

Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

Wang

Cooper

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…to Glu and Asp) measured in the presence of Ca 2ϩ are also comparable with other preparations reported so far (13), further suggesting that the N-terminal cytosolic and transmembrane domains of the enzyme are not essential for the enzymatic activity, and soluble enzyme retains the characteristic enzymatic features of the membrane-bound enzyme (32,34). Moreover, as discussed below, hydrolytic activity of sAPA toward Glu-and Asp-MCA is up-regulated comparably to native enzymes reported to date (11,13,17,35,36).…”

Section: Discussionsupporting

confidence: 82%

Enzymatic Properties of Human Aminopeptidase A

Goto

Hattori

Ishii

et al. 2006

Journal of Biological Chemistry

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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.

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“…Fourth, the cell-surface molecule specifically immunoprecipitated from pre-B-cell lines by the BP-1 antibody exhibited APA activity. Finally, APA activity detected on BP-1/6C3' cell lines exhibited the characteristic spectrum of activators and inhibitors defined for APA (18)(19)(20)(21).…”

Section: Discussionmentioning

confidence: 99%

“…The enzymatic activity was estimated by spectrophotometric measurement ofp-nitroanilide production (nmol per hr per 106 cells) from a-L-glutamyl p-nitroanilide substrates. phenanthroline; transition metal ions such as Zn2+; and competitive inhibitors such as amastatin, bestatin, angiotensin II, angiotensin III, and acidic amino acids (19)(20)(21). The APA activity of BP-1+ cells was also abrogated by these (12), also had no effect.…”

Section: Depletion Of Apa Activity From Small Intestinal Brushmentioning

confidence: 99%

Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3.

Cooper

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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The predicted amino acid sequence of the cDNA encoding the murine B-lymphocyte differentiation antigen BP-1/6C3 suggested that it is a member of the zincdependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [microsomal aminopeptidase; a-aminoacyl-peptide hydrolase (microsomal), EC 3. (refs. 4-6; unpublished results).While its broad tissue distribution implied diverse biological function, studies of the BP-1/6C3 antigen thus far have been focused mainly on its possible role in B lymphocyte development. The BP-1/6C3 molecule is expressed on pre-B and immature B-lineage cells in the bone marrow (2, 3, 7), and its expression on stromal cell lines correlates with their ability to support pre-B-cell growth (4). Moreover, interleukin 7 (IL-7) preferentially induces BP-1/6C3 expression together with pre-B-cell proliferation (6, 8) and up-regulated expression of the BP-1/6C3 antigen, which is often seen on transformed pre-B cells (1, 2, 9, 10).In an effort to define the structure and function of this interesting cell-surface molecule, we purified the antigen and cloned its cDNA from an Abelson murine leukemia virustransformed pre-B-cell line (11). The amino acid sequence predicted from the cDNA sequence suggested that the murine BP-1/6C3 antigen is a member of the zinc-dependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [APN; microsomal aminopeptidase; a-aminoacyl-peptide hydrolase (microsomal) EC 3.4.11.2]. Four distinctive types of aminopeptidases have been reported (12). These include APN, which has a relatively broad specificity acting on peptides with an N-terminal neutral amino acid; aminopeptidase A [APA; u-a-aspartyl(L-a-glutamyl)-peptide hydrolase, EC 3.4.11.7], acting on peptides with N-terminal acidic amino acids; aminopeptidase P (aminoacrylprolyl-peptide hydrolase, EC 3.4.11.9) and aminopeptidase W, which hydrolyze peptides in which the penultimate amino acid is a proline or a tryptophan.In the present studies, we have obtained evidence that the BP-1/6C3-reactive molecule exhibits APA activity.

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Purification and Characterization of Human Placental Aminopeptidase A

Cited by 40 publications

References 9 publications

Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

Enzymatic Properties of Human Aminopeptidase A

Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3.

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