Abstract
Abstract A trans-farnesyl pyrophosphate-squalene synthetase has been isolated in a soluble form from yeast extracts and purified 45-fold. The molecular weight of the enzyme estimated from sucrose density gradient centrifugation and gel filtration chromatography is 426,000. Solubilization of the squalene synthetase is achieved with deoxycholate. Treatment with the detergent markedly lowers squalene synthetase activity but when deoxycholate is removed by Amberlite XAD-2, the soluble enzyme regains full activity. Such synthetase preparations are relatively labile. They can be stabilized by glycerol and 2-mercaptoethanol. Both TPNH and DPNH serve as electron donors for the squalene synthetase. Their Km values are 122 µm and 310 µm, respectively. The two pyridine nucleotides differ somewhat in their effects on the Hill coefficient for the bimolecular condensation of farnesyl pyrophosphate to squalene. With DPNH the Hill slope is 2.0 and with TPNH 1.4. The purified synthetase catalyzes not only the formation of squalene from farnesyl pyrophosphate but also accumulates presqualene pyrophosphate (in the absence of pyridine nucleotide) and converts biosynthetic presqualene pyrophosphate to squalene.
Highlights
When DOC-treated enzyme was passed over Amberlite XAD-2, a resin that effectively absorbs hydrophobic substances including deoxycholate, the effluent was enzymatically active and to a degree which depended critically on the DOC concentration during solubilization
Under optimal conditions (0.3% DOC) squalene synthetase activity was fully recovered from the XAD-2 column
It is of interest that enzyme not treated with DOC is recovered from the XAD-2 resin in very low yield, presumably because it is tightly associated with hydrophobic membrane fragments which are bound to the resin
Summary
For testing the effect of lipids on squalene synthetase activity, Farnesyl Pyrophosphate-Squabne Absorbance of the various fractions at 280 nm (O-O), squalene synthetase activity (C-O), and (- - -) NaCl gradient, 2 ml of an enzyme preparation were extracted with chloroformmethanol according to Folch et al [14]. In extracts prepared by French pressure cell disruption of bakers’ yeast, squalene synthetase activity is partitioned between the 105,000 X g sediment and supernatant fractions.
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