A procedure is presented for the purification of alpha2u globulin, a protein synthesized in the liver, secreted into the serum, and excreted in the urine of male rats. The steady-state levels of alpha2u globulin in the serum and liver cytosol fraction of adult male rats have been determined using a radial immunodiffusion assay. A cell-free protein synthesizing system, easily prepared from commercial wheat germ, has been used to identify and quantitate the mRNA coding for alpha2u globulin. Poly(A)-containing RNA isolated from male rat liver directs the synthesis in the wheat germ translational system of a protein which is precipitated by rabbit anti-alpha2u globulin and migrates with authentic alpha2u globulin on sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-containing RNA from the livers of female rats or from the kidneys of male rats, tissues which synthesize no alpha2u globulin, do not direct the synthesis of alpha2u globulin in the wheat germ system. Thyroidectomized male rats had no detectable alpha2u globulin in their sera or liver cytosols, and the livers from these thyroidectomized males were found to contain no translatable mRNA for alpha2u globulin, as measured in the wheat germ system. Administration of L-thyroxine or triiodo-L-thyronine to hyroidectomized males resulted in the synthesis of alpha2u globulin, as measured by increased levels of this protein in sera and liver cytosols. This increase in alpha2u globulin synthesis following thyroid hormone treatment was accompanied by a parallel increase in the functional level of the hepatic mRNA coding for this protein. Treatment of thyroidectomized males with a variety of androgens failed to stimulate alpha2u globulin synthesis, and no alpha2u globulin mRNA could be detected in the livers from these androgen-treated thyroidectomized males. These findings indicate that thyroid hormones influence alpha2u globulin synthesis in male rat liver by acting pretranslationally, possibly by modulating gene transcription, and rule out the possibility of an indirect androgen-mediated effect of thyroid hormones in modulating alpha2u globulin biosynthesis.
The messenger RNA rat alpha2u globulin has been identified and quantitated in a cell-free translational system derived from Krebs II ascites cells. Hepatic tissue of the mature male rats which normally produce alpha2u globulin was also found to contain a high level of alpha2u mRNA. Approximately 1.6 per cent of all poly(A) containing RNA of the adult male rat liver could be accounted for alpha2u messenger activity. Female rats do not produce alpha2u globulin and no alpha2u mRNA activity could be detected in the poly(A) containing RNA fraction obtained from the livers of these animals. However, androgen treatment to spayed female rats was found to induce the parallel appearance to both alpha2u globulin and its corresponding mRNA. Both hypophysectomy and adrenalectomy which are known to reduce the level of alpha2u globulin in the urine of male rats were found also to reduce the hepatic level of alpha2u mRNA. The results indicate that hormonal control of alpha2u globulin synthesis in rat liver is achieved primarily through regulation of its translatable mRNA level and that more than one hormone may participate in this regulation.
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