Regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and protein mass in the ocular lens.
We have attempted to map the regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme activity and protein mass along the radial axis of the ocular lens. Because lens plasma membrane is extremely rich in cholesterol and the lens must synthesize this cholesterol, the activity of HMGR could be a key factor controlling lens membrane formation. Lenses of young rats were divided into discrete fractions by gradual dissolution in a tergitol-containing buffer; each fraction was then equated to a specific arc of the radius based upon its protein content. Aliquots of each fraction were assayed for HMGR enzyme activity and protein mass. Relative protein mass was quantitatively estimated by Western blotting using a monoclonal antibody to HMGR with immunoreactivity detected by enhanced chemiluminescence. Lens HMGR possessed a molecular mass of about 97 kDa and localized in the cell's insoluble fraction. Peak levels of both HMGR enzyme activity and protein mass were found in the outer 5% of the lens radius; levels of both decreased precipitously from there to the outer 10% radius mark. This distribution paralleled synthesis of the membrane's cholesterol, phospholipid, and intrinsic protein. We conclude that the abrupt cessation of plasma membrane synthesis in the ocular lens could involve loss of HMGR activity over a narrow arc of the lens radius, and that this activity loss is due to disappearance of enzyme protein.
Shi
H
H
Department of Biochemistry, Kirksville College of Osteopathic Medicine, MO 63501.
Cenedella
R J
RJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000032:analysis*; D007124:Immunoenzyme Techniques; D007908:Lens, Crystalline / Q000201:enzymology* / Q000648:ultrastructure; D008163:Luminescent Measurements; D008970:Molecular Weight; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
D000911:Antibodies, Monoclonal; D006903:Hydroxymethylglutaryl CoA Reductases