Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35).

The metabolism of high-density lipoprotein-associated cholesteryl esters (HDL-CE) in liver cells is not well understood. We studied the possible role of lysosomal and extralysosomal pathways on such metabolism by measuring the uptake and hydrolysis of HDL-CE in H-35 rat hepatoma cells. Incubation of cells with [3H]cholesteryl ester-labeled HDL led to the intracellular accumulation of both 3H-free cholesterol and [3H]cholesteryl ester. The ratio of 3H-free cholesterol/[3H]cholesteryl ester increased with an increase in incubation time even in the presence of chloroquine. Because chloroquine did not inhibit the conversion of cholesteryl ester to free cholesterol, the hydrolysis of HDL-CE may have been catalyzed by an extralysosomal enzyme, perhaps by neutral cholesteryl ester hydrolase (NCEH). When we incubated cells with increasing concentrations of HDL, NCEH activity increased. This increase in enzyme activity was not inhibited by the addition of chloroquine. A complex of dimyristoylphosphatidylcholine (DMPC)/apo HDL/cholesteryl ester enhanced the activity as well as native HDL. Neither the DMPC/apo HDL nor the DMPC/cholesteryl ester complex affected the activity, suggesting that apo HDL may be required for the uptake of HDL-CE. The present study demonstrated that the extralysosomal hydrolysis by NCEH is operating in the metabolism of HDL-CE in hepatoma cells.

[Askostatin--a fungicide from Streptomyces viridovulgaris]. / Askostatin--fungitsid iz Streptomyces viridovulgaris.

Askostatin is a fungicide isolated from the culture of Streptomyces viridovulgaris. It was separated by the countercurrent distribution procedure into components A and B. By the mass and NMR spectra and the biological findings the components were identified with cycloheximide and isocycloheximide respectively.

High-pressure liquid chromatographic determination of cycloheximide in ointment and suspension formulations.

A high-pressure liquid chromatographic procedure is described for the assay of the antibiotic cycloheximidein bulk drug and two experimental formulations. The method utilizes a reversed-phase C18 chromatographic column and refractive index detection. Possible impurities or degradation products, isocycloheximide, anhydrocycloheximide, and dimethylcyclohexone, are well separated from cycloheximide by this procedure. Complete extraction of cycloheximide from the formulations was obtained. The assay has a relative standard deviation of approximately 1%.