Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of 3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity 3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.