Solubilization of a particulate UDP glucose: sterol β glucosyl transferase in developing cotton fibers and seeds and characterization of steryl 6 acyl D glucosides

A particulate enzyme fraction from developing cotton fibers catalyzed the transfer of [14C]glucose from UDP-[14C]glucose to endogenous sterol acceptors, and in turn catalyzed the esterification of the steryl glucosides with fatty acids from an endogenous acyl donor. Analysis of the products by gas-liquid chromatography and mass spectrometry revealed that β-sitosterol was the predominant sterol moiety, while campesterol, cholesterol, and stigmasterol were present in smaller amounts. Glucose was the only sugar present, and it appeared to have the β-configuration. Palmitate and oleate were the major acyl components of the esterified steryl glucoside, and these fatty acid moieties appeared to be linked to the 6-position of glucose as indicated by both periodate cleavage and permethylation studies of the esterified steryl glucoside. When the UDP-glucose: sterol glucosyl transferase was solubilized with Triton X-100 and partially purified, it demonstrated an absolute requirement for added sterol acceptor and could utilize both cholesterol and stigmasterol almost as efficiently as β-sitosterol. However, it was specific for UDP-glucose and showed only slight activity with ADP-glucose, GDP-glucose, CDP-glucose, TDP-glucose, UDP-galactose, and UDP-mannose. The reaction rate was stimulated three-fold by the addition of plant lecithins, while egg lecithin had no effect. Intracellular localization studies demonstrated that the membrane vesicles containing the UDP-glucose:sterol glucosyl transferase had a sucrose density gradient profile which was similar to that of several other enzymes thought to be involved in the synthesis of cell wall polysaccharides. © 1974

Biosynthesis and structure of glycosyl diglycerides, steryl glucosides, and acylated steryl glucosides

A particulate enzyme fraction from Mycobacterium smegmatis catalyzed the transfer of14C-glucose from UDP-14C-glucose into neutral glycolipids. The two major radioactive components were purified by column chromatography on 0-diethylamino ethyl cellulose (acetate) and thin layer chromatography on silica gel in several solvents. The first product yielded a water-soluble component upon saponification, which had a hexoseglycerol ratio of 1:1 with all of the hexose being identified as glucose. The second product yielded a water-soluble component upon saponification which contained hexose and glycerol in a 2:1 ratio and, in addition to glucose, contained lesser amounts of mannose and galactose. Palmitate and oleate were the predominant fatty acids and were present in equimolar amounts. The products thus have been identified as monoglycosyldiglyceride and diglycosyldiglyceride. The diglycosyldiglyceride could also be labeled with14C-galactose when UDP-14C-galactose served as the donor, but the monoglycosyldiglyceride was only slightly labeled with14C-galactose. Membrane fractions from mung bean seedlings catalyzed the transfer of14C-glucose from UDP-14C-glucose into a neutral glycolipid which has been purified by thin layer chromatography and analyzed by combined gas liquid chromatographymass spectrometry. It was determined to be a steryl glucoside with the two major sterol components being β-sitosterol and stigmasterol linked to β-D-glucose. Particulate fractions from developing cotton fibers also catalyzed the formation of steryl glucosides and, in addition, they catalyzed the esterification of steryl glucosides at the 6 position of glucose with fatty acids (primarily palmitate and oleate) from an endogenous acyl donor. Both the glucosyl transferase and the acyltransferase have been solubilized with Triton X-100 and partially purified by chromatography on Sephadex G-200. The acyltransferase activity was reconstituted by the addition of the steryl glucoside and a phospholipid acyl donor. © 1975 American Oil Chemists\u27 Society