PURIFICATION OF A SUPERNATANT FACTOR THAT STIMULATES AMINO ACID TRANSFER FROM SOLUBLE RIBONUCLEIC ACID TO PROTEIN

The reactions involved in the transfer of amino acids from soluble ribonucleic acid (sRNA) to protein are in need of clarification, and it would be of obvious advantage in studying this transfer to work with as clean a system as possible, both in regard to the particle preparation and in regard to any soluble components involved. A major effort in the Lipmann laboratory? has been directed toward identifying and purifying the factors in the 105,OOO g supernatant fraction that stimulate amino acid transfer.' The initial observation by Hoagland et a1.2 of a stimulating effect of the pH-5 fraction on the transfer reaction in the rat liver microsomal system suggested that an enzyme or group of enzymes might be involved. Hiilsmann and Lipmann? however, observed that sulfhydryl compounds, for example glutathione or cysteine, could partially replace the 105,OOO g supernatant in stimulating the transfer of leucine from sRNA to the protein of rat liver microsomes. In many experiments glutathione and supernatant gave nearly the same degree of stimulation. I t was therefore concluded that the effect of supernatant was due, in part, to its content of glutathione, glutathione reductase, and a TPNH-generating system. The clear recognition of a sulfhydryl requirement simplified the task of recognizing other, possibly enzymic, factors involved. Although the addition of supernatant often had little effect beyond that of sulfhydryl compounds in our microsomal system, my associates and I still suspected that additional factors were involved. If so, however, the microsomes must have contained sufficient amounts to give a near-maximal rate of transfer. To probe further into this possibility deoxycholate-extracted microsomes were tried, for Kirsch4 a t the Rockefeller Institute and Korner6 at Cambridge University, Cambridge, England had found that deoxycholatetreated liver microsomes retained the ability to incorporate amino acids. Our procedure was as follows: rat liver microsomes were prepared in Littlefield and Keller'se medium A, using 24-hour fasted animais in order to deplete liver glycogen. The microsomes were well homogenized in 0.5 per cent deoxycholate and recentrifuged a t 105,OOO g for 2 hours. The pellets were rinsed several times with medium A and homogenized in the same medium. The specific activity of this preparation was about 1% times that of the original microsomes. This preparation withstood lyophilization and storage at 20" C. for as long as 3 weeks with little or no loss of activity. In contrast to untreated microsomes, the deoxycholate-extracted particles showed an absolute requirement for the 105,OOO g supernatant, even in the presence of glutathione, and the deoxycholate wash was found to contain a