Preparation of functional acetyl-CoA carboxylase mRNA from rat mammary gland

Abstract

Poly(A)+ RNA from lactating rat mammary glands was fractionated according to size by isokinetic sucrose gradient centrifugation to obtain a fraction enriched for acetyl-CoA carboxylase. In vitro translation of this RNA preparation yielded apparent full-length acetyl-CoA carboxylase with a molecular weight of 260,000. The synthesized protein was identified as acetyl-CoA carboxylase by specific immunoprecipitation. Tests with antiserum to fatty acid synthetase, revealed that the fractions containing acetyl-CoA carboxylase mRNA also contained mRNA for fatty acid synthetase; both of these mRNAs were approximately 10 kb. Fatty acid synthetase with a molecular weight of 250,000 was synthesized. Using an in vitro rabbit reticulocyte lysate translation system, we have shown that the amount of translatable acetyl-CoA carboxylase mRNA increases during lactation. On the fifth day postpartum the level of translatable acetyl-CoA carboxylase mRNA increased to a peak level seven times that on the day of parturition.