1). As judged by the SDS-PAGE data of Fig. 3C, lane 4, transferrin is the only contaminant of our CPA2 preparation obtained from rat MAB perfusate; no attempts were made to rid the CPA2 preparation of this proteolytically inactive substance for the sake of recovery of CPA

activity during the purification process. The finding of a functional CPA2 in the rat MAB perfusate, an enzyme which structurally resembles that isolated from pancreas [10], led us to investigate the presence of the respective RNA message www.selleckchem.com/products/PF-2341066.html in the rat mesentery as an evidence of the local synthesis of the enzyme. The complete sequence of the cloned cDNA for the rat mesenteric preproCPA2 was obtained as described in Section 2.6.2, which turned out to be identical with that

of rat pancreas [10] except for the base changes G177T, C439A and C1148G. The latter two changes introduced the mutations selleck chemical L147I and A383G (preproCPA2 numbering), indicating the polymorphic nature of this enzyme. Whereas the rat MAB perfusate CPA1 was isolated as an Ang-(1-7)-forming enzyme using Ang II as the substrate for monitoring the activity during the chromatographic purification of the enzyme (Fig. 1A), the isolation of the CPA2 was attained by measuring its activity toward Z-Val-Phe, a general purpose substrate for CPAs (Fig. 3A and B). In spite of this, CPA2 was later shown capable of hydrolyzing some Ang peptides with substrate specificity suggestive that differences between rat MAB CPA1 and CPA2 go beyond those observed during their isolations. These enzymes

differentially process peptide components of the RAS such as Ang I, Ang II, Ang-(1-9) and Ang-(1-12), as shown in Fig. 5 and Fig. 6. For the sake of comparison between enzyme preference for different substrates, hydrolysis of each of these peptides was determined using a fixed Tau-protein kinase amount of either enzyme (0.41 mU CPA1 and 1.02 mU CPA2, based on Z-Val-Phe hydrolysis), chosen so as to limit the cleavage of the substrate that was most rapidly degraded by the respective enzyme to about 80% of its initial concentration. Rat CPA1 is remarkable in which, under the assay conditions described in Fig. 5A, it is capable of generating Ang-(1-7) by a stepwise mechanism involving the sequential removal of the C-terminal residue of the intermediate products Ang-(1-9) and Ang II. This mechanism is rather corroborated by the results of the direct action of rat CPA1 on the substrates Ang II and Ang-(1-9), shown in Fig. 5B and C, respectively. Moreover, the accumulation of the intermediate Ang-(1-9) during the conversion of Ang I to Ang-(1-7) suggests that the cleavage of the C-terminal His residue of Ang-(1-9) may be the limiting step in the process. On the other hand, rat CPA2 generates only Ang-(1-9) upon incubation with Ang I (Fig. 6A) and, under the reaction conditions described in Fig. 6B and C, has a negligible, if any, action on Ang II and Ang-(1-9), respectively.