Fatty alcohol synthesis in mouse preputial glands by microsomal acyl-CoA reductase
Date Issued
December 1, 1980
Author(s)
Moore, Claudia
Advisor(s)
Fred Snyder
Additional Advisor(s)
Ten-Ching Lee
Francis T. Kenny
Stephen J. Kennel
Jeffery M. Becker
Abstract
The synthesis of fatty alcohols in preputial glands of mice was shown to be catalyzed by an NADPH-dependent enzyme that reduces palmitoyl-CoA to hexadecanol. This acyl-CoA reductase was located exclusively in the microsomal fraction, specifically on the cytoplasmic side of the membrane. The reaction was not catalyzed by either a nonspecific alcohol dehydrogenase or an aldehyde reductase. The reductase was sensitive to sulfhydryl and serine reagent modification, had a pH optimum of 6.8, and produced an aldehyde intermediate. Acyl-CoA reductase was detergent-sensitive and the activity could not be restored after removal of the detergents. Phospholipase C and asolectin did not release acyl-CoA reductase from the microsomal membrane but did cause a significant decrease in the activity recovered in the membrane pellet. Glycerol did not solublize any of the reductase activity, nor did 3.0 M NaCl; however, the combination of glycerol and 3.0 M NaCl did release about 50% of the acyl-CoA reductase from the microsomal membranes. Expression of the enzyme activity required certain phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved by a modification of lipid environment where it resides. Substrate concentration curves in the presence and absence of bovine serum albumin demonstrated significant alterations in enzyme activity. The reductase was very sensitive to the concentration of palmitoyl-CoA and was progressively inhibited beyond the critical micellar concentration of the substrate. In the presence of various albumin concentrations, the apparent Km for acyl-CoA reductase was 14 µM; the maximum velocity varied with the concentration of albumin. A long chain acyl-CoA binding protein (Z protein) located in the cytosol of preputial glands stimulated acyl-CoA reductase activity to almost the same extent as did albumin. The cytoplasmic acyl-CoA binding protein was stable to heating and lyophilization but was destroyed by trypsin treatment. It bound palmitoyl-CoAs and eluted from a Sephadex G-100 column at the same position as the Z protein isolated by others from liver and intestinal mucosa. Based on gel elution behavior, the molecular weight of the stimulatory protein in the preputial gland cytosol was estimated to be between 10,000 and 12,000. During puberty, the preputial gland differentiates into a functional gland under the influence of testosterone. The level of acyl-CoA reductase activity was shown to correlate with this differentiation; the enzyme activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. Then by 8 weeks, the activity had decreased to that found in mature male mice as sexual maturity was reached. In addition, the cytosol from the preputial glands of the youngest mice (3 weeks) contained enough Z protein to stimulate acyl-CoA reduction. However, the 3-week old preputial gland microsomes contained little or no acyl-CoA reductase. As the animal matured, stimulatory capacity of the cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction leveled off. Therefore, it appeared that acyl-CoA reductase activity and the levels of Z protein had similar patterns of development as the preputial gland during puberty. Results of this study indicate that the regulation of fatty alcohol synthesis, in vivo, is controlled by the levels of acyl-CoAs, Z protein, and acyl-CoA reductase activity.
Degree
Doctor of Philosophy
Major
Biomedical Sciences
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