CHEMICAL AND ENZYMATIC INVESTIGATIONS OF THE MECHANISM OF THE REMOVAL OF THE 14ALPHA-METHYL GROUP OF LANOSTEROL

A variety of 14(alpha)-methyl, 14(alpha)-hydroxymethyl, and 14(alpha)-hydroxy sterols were prepared by chemical synthesis for use in investigations of the mechanism of the enzymatic removal of the 14(alpha)-methyl group (carbon atom 32) of lanosterol during the biosynthesis of cholesterol. A comparison of the in vitro metabolism of {2,4-('3)H}14(alpha)-hydroxymethyl-5(alpha)-cholest-7-en-3(beta)-ol (I) and {2,4-('3)H}14(alpha)-hydroxymethyl-5(alpha)-cholest-7-ene-3(beta),15(alpha)-diol (II) was made using 10,000 x g supernatant fractions of liver homogenates from male rats. Compound I was found to be efficiently converted to tritium-labeled cholesterol upon aerobic incubation with these enzyme preparations supplemented with NAD and NADP; in the absence of a cofactor supplement, a variety of other labeled C(,27) monohydroxy sterols, in addition to cholesterol, were isolated and characterized, including 5(alpha)-cholest-8(14)-en-3(beta)-ol, 5(alpha)-cholest-7-en-3-ol, 5(alpha)-cholesta-8,14-dien-3(beta)-ol, and cholesta-5,7-dien-3(beta)-ol. Compound II, which differed from I only by the addition of a 15(alpha)-hydroxyl group, was not significantly metabolized to cholesterol in either the presence or the absence of a cofactor supplement. These results do not favor the intermediary role of 15(alpha)-hydroxy sterols during the enzymatic removal of carbon atom 32 of 14(alpha)-methyl sterols. Incubation of compound I with a washed microsomal preparation (derived from the livers of male rats) supplemented with NAD and an NADPH generator under aerobic conditions yielded cholesterol and 5(alpha)-cholest-7-en-3(beta)-ol as the principal labeled C(,27) monohydroxy sterol products. However, incubation of I with washed microsomes under aerobic conditions in the absence of added cofactors or in the presence or absence of supplemental NAD and NADP under anaerobic conditions yielded 5(alpha)-cholest-8(14)-en-3(beta)-ol as the major ((GREATERTHEQ) 90%) labeled C(,27) monohydroxy sterol product. The extent of conversion of I to C(,27) monohydroxy sterols was roughly the same in all of the microsomal incubations, regardless of the presence or absence of oxygen and/or a cofactor supplement. These results indicate that oxygen is not required for the enzymatic removal of carbon atom 32 of 14(alpha)-hydroxymethyl-5(alpha)-cholest-7-en-3(beta)-ol. It is further suggested by these results that oxygen is required only for the initial hydroxylation of the 14(alpha)-methyl group during the removal of carbon atom 32 of lanosterol, and that the immediate product of this demethylation process is a sterol containing a (DELTA)('8(14)) double bond. Washed microsomal preparations were also found to catalyze, under anaerobic conditions, the isomerization of 14(alpha)-methyl-5(alpha)-cholest-7-en-3(beta)-ol and 14(alpha)-hydroxymethyl-5(alpha)-cholest-7-en-3(beta)-ol to 14(alpha)-methyl-5(alpha)-cholest-8-en-3(beta)-ol and 14(alpha)-hydroxymethyl-5(alpha)-cholest-8-en-3(beta)-ol, respectively. Two sterols with the "unnatural" cis-C-D ring junction were prepared in labeled form: {2,4-('3)H}5(alpha),14(beta)-cholest-7-en-3(beta),15(beta)-diol (III) and {2,4-('3)H}5(alpha),14(beta)-cholest-7-en-3(beta),15(beta)-diol (IV). Compound III, but not its 15(beta)-hydroxy epimer (IV), was found to be efficiently converted to cholesterol in 10,000 x g supernatant fractions of liver homogenates from male and female rats. After the incubation of III, a number of other labeled products, in addition to cholesterol, were isolated and characterized, including 5(alpha)-cholest-8-en-3(beta),ol, 5(alpha)-cholest-7-en-3(beta)-ol, 5(alpha)-cholesta-8,14-dien-3(beta)-ol, and cholesta-5,7-dien-3(beta)-ol.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Natural Sciences

Grantor

Rice University

Year dc:date.issued

1980

Author dc:creator

• PASCAL, ROBERT ANTHONY, JR.

• Biochemistry

Statement dc:rights

• Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1911/15576

OAI identifier oai:identifier

oai:repository.rice.edu:1911/15576