Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum

A modification of the method of assay of it-amylase described by P. Bernfeld (2) which measures the release of dextrins, or maltose, from a soluble starch substrate at 37°C, was used to assay the enzymatic activity of lysates from COS7 cells which had been transfected with plasmids containing the wildtype a-amylase or the Al-I4m/Am chimera. Briefly, 100-mm dishes of COS7 cells, 48 h post-transfection, were rinsed free of medium and secreted products with PBS, scraped and then lysed in 1× lysis buffer (31) . Volumes of 50, 1130, 175, or 200 I11 of lysate were each diluted to a final volume of 250 ~tl with a buffer containing 20 mM sodium phosphate, 20 mM NaCI, 0.2 % Triton X-100, and placed on ice. After the addition of 250 I.tl of a 1% soluble starch substrate made up in the same buffer, to each sample or control tube, the samples were incubated at 30°C for 15 min before being placed on ice. One ml of a color reagent, consisting of 1% 3,5-dinitrosalicylic acid in 0.2 M potassium hydroxide and 0.021 M sodium potassium tartrate was added, each was boiled for 25 min, placed on ice, and then diluted with 3.5 ml of water. The optical density of the samples was read at 520 nm and the values compared against a maltose standard curve prepared from 0, 5, 10, 25, 50, and 100-1~1 aliquots of a 10 mg/ml maltose stock solution. An a-amylase standard curve was obtained by using aliquots of a-amylase (Sigma Chemical Co.) in the identical assay procedure.