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Isolation and characterization of Brush border fragments from mosquito mesenterons
Author(s) -
Houk Edward J.,
Arcus Yvonne M.,
Hardy James L.
Publication year - 1986
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.940030204
Subject(s) - brush border , biology , aminopeptidase , isoelectric focusing , alkaline phosphatase , biochemistry , esterase , leucine , papain , enzyme , microbiology and biotechnology , staining , vesicle , amino acid , membrane , genetics
Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis , by a combination of Ca 2+ precipitation and differential centrifugation. These preparations were routinely enriched seven‐ to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane‐bound enzyme. The predominant nonspecific esterase isozyme remained membrane‐bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two‐dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.