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Aminopeptidase a from Rhynchosciara americana (Diptera) larval midguts: Properties and midgut distribution
Author(s) -
Klinkowstrom Adriana M.,
Terra Walter R.,
Ferreira Clélia
Publication year - 1994
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.940270406
Subject(s) - aminopeptidase , biology , midgut , biochemistry , enzyme , leucyl aminopeptidase , size exclusion chromatography , glycocalyx , differential centrifugation , hydrolase , microbiology and biotechnology , leucine , amino acid , larva , botany
L‐aspartic acid α‐(β‐naphthylamide) (AspβNA) 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 AspβNA 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 AspβNA well and displays a low activity on LpNA and proline β‐naphthylamide (ProβNA). Thus, this enzyme is an aminopeptidase A (EC 3.4.11.7). It has a pH optimum of 7.5, M r 117,000 (gel filtration), and is competitively inhibited by aspartate hydroxamate (K i 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. © 1994 Wiley‐Liss, Inc.