No abstract

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca2+ 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. lsoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [I], and alkaline phosphatase [ I ] 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.

A purified enzyme mixture obtained by extraction of Aspergillus niger cells with saline was shown to have glycosidase, carboxypeptidase, and endopeptidase activities and to abolish the hemagglutinating and infectious activities of the PR8 strain of influenza virus. The endopeptidase activity of the A.niger extracts was shown to be responsible for inactivation of the virus.

The PR8 strain of influenza A virus was grown in canine kidney cell cultures and in chick and duck embryos. After purification by density gradient procedures, the virus preparations were tested for the presence of host antigens with antisera to appropriate host materials. All PR8 preparations were shown to have serologically detectable host components. These host antigens appeared not to be closely associated with the hemagglutinin or neuraminidase spikes, since virus from which essentially all of these structures were removed formed substantial precipitates with antiserum to host material. Evidence is also reported which suggests that cellular actin is an integral component of highly purified PR8 virions, though probably unrelated to host antigen.