Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization

Abstract

Glutamyl aminopeptidase [aminopeptidase A (EAP), EC 3.4.11.7] is an ectoenzyme that selectively hydrolyzes acidic amino acid residues from the amino terminus of oligopeptides. EAP activity is highest within the kidney and small intestine. The murine pre-B cell BP-1/6C3 and the human kidney glycoprotein gp160 differentiation antigens have been reported to have biochemical properties indistinguishable from EAP. It is not known, however, if rat kidney EAP is a homologue of these antigens or molecularly distinct. Using the reverse transcription-polymerase chain reaction method with oligonucleotide primers based on the BP-1/6C3 nucleotide sequence, we isolated a 450-bp partial cDNA from rat kidney poly(A)+ RNA. The partial cDNA encoded a predicted protein that was 92% and 86% identical to the murine BP-1/6C3 and human gp160 antigens, respectively; the amino acid sequence within the zinc-binding domain was completely conserved. Purification of EAP from rat kidney and microsequence analysis of a tryptic digest peptide fragment (18-mer) indicated that the fragment was highly similar to a region within the BP-1/6C3 and gp160 proteins. Northern blot hybridization and immunoblot analyses were also consistent with labeling of products the same size as reported for the BP-1/6C3 and gp160 antigens. There was a good correlation between the cellular distribution of EAP mRNA and EAP immunoreactivity, with proximal tubules and glomerular mesangial cells having the highest densities. These results indicate that rat kidney EAP is a species homologue of the murine BP-1/6C3 and human gp160 antigens. Furthermore, on the basis of its cellular localization, rat kidney EAP is likely to be involved in degradation of oligopeptides within the glomerulus and the glomerular filtrate. Since cells that express EAP also express receptors for angiotensin II, an intrarenal vasoactive hormone that is a substrate for EAP, these results further suggest that EAP may play a role in modulating the activity of intrarenal angiotensin II.

Kidney Aminopeptidase A and Hypertension, Part I

“…37 Briefly, 16-week-old SHR and WKY were killed and their kidneys were immediately removed and placed in ice-cold D-PBS, pH 7.4, containing (in mmol/L): NaCl 137, KCl 2.7, Na 2 HPO 4 8.1, KH 2 PO 4 1.5, CaCl 2 0.9, MgCl 2 0.49, and glucose 5.6. Six kidneys from separate animals were pooled for isolation of glomeruli.…”

“…37 APA activity was measured by use of ␣-glutamyl-2-naphthylamide (Bachem Bioscience) as substrate. Enzyme-specific activity was expressed as mol/mg protein per hour.…”

“…37,41 Briefly, samples were separated on a 10% SDS-PAGE gel and transferred to an Immobilon membrane (Millipore). The membranes were washed and incubated with primary antiserum (1:3000 dilution) overnight at 4°C.…”

“…36 The highest levels of APA are found in the kidney and small intestine, with lower levels of APA associated with vascular elements found throughout the body. In the kidney, APA is expressed in proximal tubule brush border, 37 and glomerular mesangial, and epithelial cells. [37][38][39][40] Therefore, in the present study, we sought to determine whether tissue and plasma APA levels are altered during the development of hypertension in SHR compared with its normotensive control strain, WKY.…”

“…In the kidney, APA is expressed in proximal tubule brush border, 37 and glomerular mesangial, and epithelial cells. [37][38][39][40] Therefore, in the present study, we sought to determine whether tissue and plasma APA levels are altered during the development of hypertension in SHR compared with its normotensive control strain, WKY. Our findings indicate that the major difference between strains seems to be elevated activity of APA within SHR kidney at all stages of hypertension.…”