Aminopeptidase A from Rhynchosciara americana (Diptera) larval midguts: properties and midgut distribution
1994
Klinkowstrom, A.M. | Terra, W.R. | Ferreira, C.
L-aspartic acid alpha-(beta-naphthylamide) (Asp beta NA) hydrolase activity is restricted mostly to the midgut caeca of Rhynchosciara americana larvae. The membrane-bound activity is solubilized in detergent and, after electrophoretic separation, proved to be identical to leucine p-nitroanilide (LpNA) hydrolases previously described. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and aminopeptidase, suggests that the soluble Asp beta NA hydrolase is associated with the cell glycocalyx. Soluble aminopeptidases from R. americana midgut caeca are resolved into three fractions by gel electrophoresis. The slow migrating fraction hydrolyzes Asp beta NA well and displays a low activity on LpNA and proline beta-naphthylamide (Pro beta NA). Thus, this enzyme is an aminopeptidase A (EC 3.4.11.7). It has a pH optimum of 7.5, Mr 117,000 (gel filtration), and is competitively inhibited by aspartate hydroxamate (Ki 0.1 mM). Nevertheless, this enzyme, in contrast to the vertebrate enzyme, is not activated by calcium ions. The aminopeptidase A seems to have a charge variant that displays an intermediate migration and is not resolved from an aminopeptidase N (enzyme very active on LpNA). These two activities are not resolved by either gel filtration or ion-exchange chromatography. The aminopeptidases N with intermediate and high migration, previously reported to be charge variants, were shown in this paper to differ in substrate specificities and in the strength with which they associate to the cell glycocalyx.
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