| Abstract: | 1. The effect of growth status on the relative levels and recoveries of DNA-dependent RNA polymerase in rat liver nuclei was determined by two independent procedures: (a) measurement of RNA polymerase A and B activities in fraction IV [Roeder, R. G. and Rutter, W. J. (1970) Proc. Natl Acad. Sci. U.S.A. 65, 675--682] in the presence and absence of low concentrations of alpha-amanitin; (b) DEAE-Sephadex chromatography of fraction IV to resolve RNA polymerases A and B (and possibly other forms of the enzyme). 2. Growth was arrested in young rats (less than 100 g body weight) by hypophysectomy and stimulated by the administration of growth hormone or triiodothyronine. Under these conditions the rate of RNA synthesis in vivo or in isolated nuclei is known to be markedly depressed or stimulated relatively soon after hypophysectomy or hormone administration, respectively. RNA polymerases were obtained from animals under different growth conditions. There were no differences in the activities of nuclear RNA ploymerases per se, when these were separated from their endogenous template and assayed with heterologous denatured DNA. These reports contrast with earlier reports [Smuckler, E. A. and Tata, J. R. (1971) Nat. New Biol. 234, 37--39; Sajdel, E. M. and Jacob, S. T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715]. 3. The discrepancy was resolved when a 'balance sheet' of enzyme recovery was established. Cessation of growth by hypophysectomy led to a marked reduction in the recovery of both forms A and B of the enzyme (less than 20% of the input RNA polymerase activity in fraction iv) following chromatography on DEAE-Sephadex. This effect was reversed within a short time after the administration of growth hormone (3--9 h) or triiodothyronine (18--24 h), leading to a doubling of the enzyme recoveries. These alterations which were more marked for RNA polymerase A, resulted in different elution profiles for RNA polymerases A and B upon chromatography. 4. It is concluded that the use of DEAE-Sephadex chromatography to compare the levels of RNA polymerases A and B isolated from tissues of different growth rate can give rise to over-estimates of apparent changes in their relative activities and that the measurement of enzyme activity in fraction IV is a better index of RNA polymerase levels. The relationship between growth rate of cells, the stability of RNA polymerases, and the importance of determining enzyme recoveries upon chromatography, are discussed. |
