Poly(adenylic acid) polymerase: Loss of enzyme from rat liver nuclei isolated under isotonic conditions

Abstract

Poly(A) polymerase (EC 2.7.7.19), the enzyme which appears to be responsible for the polyadenylation of mRNA, has been detected in several mammalian tissues and is present in nuclear [l-4] , mitochondrial [5-71, microsomal [8,9] and cytosol [ 10,111 fractions. We have recently purified this enzyme to homogeneity from nuclei of rat liver and a rat hepatoma [12] and from mitochondria of a rat hepatoma [ 131. Likewise, poly(A) polymerase has been purified from calf thymus tissue [ 14,151. Now that the in vitro characteristics of this enzyme have been defined, it is possible to study the response of poly(A) polymerase to various physiological parameters. Indeed, recent studies in our laboratory indicate that nuclear poly(A) polymerase levels appear to be altered in response to amino acid supply [ 161, glucocorticoid hormone [ 171 and neoplasia [12] In ordek to evaluate quantitatively the levels of enzyme in the nucleus, we determined the effect of different conditions of homogenization of liver tissue on the resultant levels of poly(A) polymerase obtained in the nuclear and cytosol fractions. In this report, we present evidence which indicates that homogenization of liver tissue with isotonic sucrose, a procedure usually employed to obtain the cytosol fraction, results in a loss of almost all the enzyme activity from nuclei. The release of the nuclear enzyme can be minimized by replacing isotonic sucrose with the hypertonic sucrose which is conventionally used for isolation of pure nuclei from mammalian tissues [18,19]. These studies demonstrate the need for the use of hypertonic sucrose in the isolation of nuclei in order to prevent loss of free nuclear enzymes.