View source for Yeast Invertase Assay

==Invertase Secretion Assays==

taken from PMID 10564282

The invertase secretion assays were performed according to the methods of Novick and Schekman (1979) with some modifications. Yeast cells were grown at 24°C to an A600 of 0.2-0.5 in YPUATD medium. Ten A600 units of cells were harvested, washed in distilled water, and induced for invertase expression by resuspension in YPUAT(low D)S medium (containing 0.05% glucose and 2% sucrose) prewarmed to 37°C. Cell samples were taken after 0, 15, 30, 45, and 60 min of induction at 37°C. Membrane transport was blocked by addition of sodium azide to 10 mM final concentration and transfer to ice. Cells of each sample were washed twice in ice-cold 10 mM sodium azide and resuspended in 2 ml of ice-cold 10 mM sodium azide. The final A600 was then adjusted to 0.5 with ice-cold 10 mM sodium azide. Two 0.5-ml aliquots of each sample were transferred to fresh microfuge tubes. To one aliquot of cells, 50 µl of distilled water were added, and the cells were left on ice (whole cells). To the other aliquot of cells, 50 µl of 10% Triton X-100 were added. Cells of this aliquot were permeabilized by freezing in liquid nitrogen and thawing at room temperature (lysates) and placed on ice. Aliquots of whole cells and lysates were assayed for invertase enzyme activity using the method of Goldstein and Lampen (1975). Ten-microliter samples were added in duplicate to 25 µl of 0.2 M sodium acetate, pH 4.9, and 12.5 µl of 0.5 M sucrose on ice. For the standard curve, known amounts of glucose (0, 12.5, 25, 50, 75, and 112.5 nmol) were used in place of the sample and sucrose. The tubes were incubated at 37°C for 10 min and then placed on ice. The reaction was terminated, and the invertase was inactivated by addition of 50 µl of 100 mM potassium phosphate buffer, pH 7, and heating to 90°C for 3 min followed by chilling on ice. After addition of 500 µl of solution C (50 µg/ml glucose oxidase [Aspergillus niger; Fluka], 10 µg/ml horseradish peroxidase [Fluka], 10 mM potassium phosphate buffer, pH 7, 300 µg/ml o-dianisidine [Sigma, St. Louis, MO], and 38% wt/vol glycerol), the samples were incubated at 30°C for 20 min. The assay was terminated, and the color was developed by addition of 750 µl of 6 M HCl. Absorbance at 540 nm was determined spectrophotometrically.

The readings for the duplicate samples were averaged. The internal invertase activity was calculated from the difference between the total invertase activity (from lysates) and the surface invertase activity (from whole cells) at each time point. The A540 values for the duplicate glucose standards were also averaged and plotted to give a glucose standard curve. The A540 values for the total, external, and internal invertase in the cell samples were then converted into nanomoles of glucose formed using the calculated slope of the glucose standard curve as a conversion factor (usually ~1 A540 unit represents 100 nmol of glucose). The internal and external invertase activity was then expressed as micromoles of glucose formed per A600 unit of cells per minute and plotted as a function of time after invertase induction. 

[[Category:Yeast]]
[[Category:Trafficking]]