Abstract
Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.
Highlights
Squalene epoxidase activity has been studied in cellfree preparations of Chinese hamster ovary (CHO)cells and rat liver
Comparison between cofactors required for activity of squalene epoxidase from CHO cells and rat liver
Dzyvential efects of PG and cytosolic fraction on microsomal epoxia'ases derivedjom CHO cells and rat liuez Rat hepatic squalene epoxidase is known to require the combination of microsomes and cytosolic supernatant fraction for full activity [1, 2]
Summary
500 ml of C H O cell suspension (ca. 1 x 10' cells) was centrifuged at 200 g for 5 min, washed with phosphate-buffered saline (PBS)and with hypotonic buffer, containing 1 mM Tris-HC1, pH 7.5, and 1 mM EGTA (buffer K). 1 x 10' cells) was centrifuged at 200 g for 5 min, washed with phosphate-buffered saline (PBS)and with hypotonic buffer, containing 1 mM Tris-HC1, pH 7.5, and 1 mM EGTA (buffer K). The cell pellet was suspended in 1.5 ml of ice-cold buffer K and homogenized in a glass Teflon homogenizer. The homogenate was centrifuged at 12,000g for 20 min at 4OC. The sediments of the corresponding centrifugation were suspended in PBS containing 0.25. Rat liver microsomes and supernatant fractions were prepared according to Yamamoto and Bloch [1] except that the working buffer was 10 mM Tris-HC1, pH 7.4, containing 0.3 M sucrose. The soluble fractions of livers from rats maintained on N-diet (NsIo5), CM-diet (CMs,,,), and S-diet (Ss,,,), as well as the corresponding microsomal preparations, were stored in liquid N P
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