Myo-inositol phosphates from beef brain phosphoinostide
Journal of Biological Chemistry
PC 23 lowed by dinitrophenylation, revealed the presence of DNPglutamine in the chromatogram, indicating a y-glutamyl-RNA. Since these two new sRNA derivatives could be intermediaries in GSH synthesis, the enzymatic synthesis of GSH was reinvestigated, largely with the use of the procedures elaborated by Konrad Bloch and his collaborators (7, 8). The finding that addition of RNase practically completely abolished GSH synthesis seemed to confirm our suspicion. In view of the isolation of the
... solation of the glutamylcysteinyl-sRNA, further exploration centered on the better characterized second phase of GSH synthesis. A partial loss of activity was obtained by treating the preparation of Snoke et al. (8) with manganous chloride according to Korkes et al. (9) ; with such a preparation, synthesis was stimulated more than 3fold by addition of either a phenol extract or the manganous precipitate. Protamine or charcoal treatment at neutral pH was ineffective, but a complete resolution of the enzyme into an RNA fraction and a protein fraction was achieved by the use of a DEAE-cellulose column. When the enzyme was placed on a column equilibrated with 0.2 M sodium phosphate buffer, pH 6.2, the column retained all RNA, and the protein fraction was collected by washing with the same buffer. The RNA was then eluted with 1 M Tris buffer, pH 7.3 and showed a 260:280 rnp absorbancy ratio of 1.83, indicating practically pure nucleic acid. The complementarity of these two fractions is shown in Table I . In contrast to peptide synthesis, hydroxamate formation with the use of 1 M hydroxylamine appeared to be independent of RNA. The coenzyme or carrier function of the sRNA was further identified by carrying out the reaction in two steps. Radioactive glutamylcysteine was derived from radioactive GSSG by hydrolysis with carboxy-peptidase and reduction of the diglutamylcystine with Zn dust.2 C14-Glutamylcysteine was incu-2 D. Strumeyer and K. Bloch, personal communication.