Ribonuclease and RNA polymerase activities in livers of rats growing at different rates.

Abstract

The activities of ribonuclease and RNA polymerase were measured in the livers of rats growing at different rates. Three weeks following hypophysectomy different growth rates were established by administration of varying doses of growth hormone over a ten-day period. Measurement of enzyme activities on the eleventh day showed that chronic administration of growth hormone caused a dose-dependent increase in RNA polymerase activity and a decrease in ribonuclease activity. {Endocrinology 89: 1120, 1971) P studies (1, 2) have revealed that ribosomes from livers of hypophysectomized (hypox) rats are normal in their ability to support poly U-directed phenylalanine incorporation, and exist in normal polyribosome arrays. The poor incorporation and degraded polysome patterns occasionally observed in preparations from hypox rat livers have been attributed to the presence of active ribonuclease observed in such preparations (1). Thus, the difference in protein synthesizing capacity in vivo between the livers of normal and hypox rats may be related to the number of ribosomes per cell rather than to a difference in their functional capacity. Cardell (3) has presented morphological evidence suggesting that there are fewer ribosomes in the liver cells of hypox rats than in those of normal rats. Biochemical evidence (4) also would tend to support this contention. If the level of protein synthesis in a tissue is regulated by the cellular concentration of ribosomes, then a mechanism must be available for modulating the ribosome population in the cells. This regulation could be effected by governing the rate of formation and/or degradation of RNA. Enzymes which are likely candidates to exert such control are ribonuclease and RNA polymerase. In the present study the activities of these two enzymes were measured in the livers of animals growing at different rates. Different growth rates were established by the administration of varying doses of growth hormone to several groups of hypox rats. After ten daily Received January 21, 1971 *NIH-GH-B14; a gift from the Endocrinology Study Section of the National Institutes of Health. injections of the hormone, the enxyme activities V of these groups were measured. Materials and Methods T Details of the experimental procedure were as follows: female Sprague/Dawley rats (90 g) obtained from Hormone Assay r Laboratories, Inc., Chicago, were maintained for three weeks on Hypophysectomized Rat Diet (5) (Nutritional Biochemicals) and then randomized into groups of four. Each animal in a group was given an intraperitoneal injection of either saline or a specific amount of bovine growth hormone* in 1 saline. The daily doses of growth hormone (GH) administered intraperitoneally in 0.50 ml were 5, 10 and 100 ug. Injections were continued for ten consecutive days. On the eleventh day, the animals were killed by decapitation and bled. Livers from the four animals in each group were removed and placed in 5 volumes of 0.25 M sucrose plus 0.05 M MgCl2. After homogenization in a Teflon-glass homogenizer, the homogenate was centrifuged first at 600 X g (6 min) and then 78,000 X g (60 min). The 78,000 X g supernate was used for the ribonuclease assay, whereas the 60 X g pellet, after redistribution in 2.4 M sucrose containing 0.001 M MgCl2, was used for isolation of nuclei according *• to the Chaveau procedure (6). The nuclear pellet obtained was used as the source of RNA polymerase which was prepared and assayed as an aggregate enzyme according to the procedure of Weiss (7). Total alkaline ribonuclease activity was measured in the presence of p-chloromercuribenzoate as described by Shortman (8). Under the conditions emu ployed 51% of the total activity of the initial homogenate was present in the 78,000 X g supernatant. Protein was assayed by the method of Lowry et al. (9), RNA by the method of Cerriotti (10), and ** DNA by the method of Burton (11); bovine serum albumin, yeast ribonucleic acid and deoxyadenosine were used as standards.