EFFECTS OF GROWTH HORMONE AND TRI-IODOTHYRONINE ON AMINO ACID INCORPORATION BY MICROSOMAL SUBFRACTIONS FROM RAT LIVER

A 2-step procedure for the separation of smooth and rough endoplasmic reticulum of rat-liver and the fractionation of the latter into "light rough membranes," "heavy rough membranes" and "free polysomes" is described. The distribution of RNA, protein and phospholipid in these fractions as well as their amino acid incorporation characteristics are also described. The heavy rough membrane fraction incorporated 14C-labelled phenylalanine, isoleucine and lysine into protein at a rate 3-5 times greater than that exhibited by the light rough membranes and free polysomes. Stimulation of the incorporation of [14C]phenylalanine and [14C]isoleucine by poly U and poly UA showed that, per mg ribosomal RNA, the heavy rough membrane fraction was also more active in its response to synthetic messenger RNAs than the other 2 fractions. Hypophysectomy caused a marked reduction in the amount of ribosomes and phospholipids, the amino acid incorporation capacity and the response to poly U and poly UA in all 3 rough microsomal subfractions. These effects were reversed in 3-4 days by a combined treatment of hypophy-sectomized rats with human growth hormone and 3,3'',5-tri-iodo-L-thyronine. Although the amino acid incorporation activity in the presence of endogenous and synthetic template RNA was affected in all the subfactions, the effect of hormone deprivation and replacement was most marked in the heavy rough membrane fraction. The relative stimulation of the incorporation of [14C]phenylalanine and [14C]iso-leucine by poly U and poly UA, respectively, by free and membrane-bound polysomes was only slightly lowered in a few cases after hormonal treatment of hypophysectomized rats. However, per mg ribosomal RNA, hormone treatment led to a 2[long dash]4-fold enhancement of the incorporation of phenylalanine and isoleucine in response to the synthetic polyribonucleotides. Treatment of heavy rough membranes with Na deoxycholate showed that much of the hormonal effect on amino acid incorporation was manifested in the ribosomes but that the maximal hormonal effect was detected when these were associated with the membranes. Growth hormone and thyroid hormone together control both the amount and amino acid incorporation activity of free and membrane-associated polyribosomes of rat-liver.