4-43?
COMMUNICATIONS TO TIIE EDITOK
however, a small but definite amount of enzymatic activity even when special precautions were taken to eliminate DPGA from the reaction. This activity was ascribed to an activated form of the enzyme, probably a phosphorylated form. Another possibility was that cofactor DPGA was tightly bound to the enzyme and was therefore always present. I n order to differentiate between these two alternatives, use has been made of Ci4and P32-labeledsubstrates. Carboxyl-labeled 3-phosphoglyceric acid (3PGA) was obtained by treating ribulose 1,5-diphosphate with C14-sodium bicarbonate in the presence of purified carboxydismutase as described by Calvin, et a1.' To a solution containing 0.17 Hmole of CI4-3PGA, 0.15 ymole DPGA and 50 pmoles of imidazole-HC1 buffer pH 7.0 was added 0.2 pmole of crystalline enzyme (final volume 1.5 ml.). two hours of incubation a t 30' the solution v a s dialyzed extensively against several changes of distilled water and sodium chloride solution. The final dialysate possessed no radioactivity but the protein solution was radioactive, and the amount of radioactivity in this solution indicated that there was about one mole of phosphoglyceric acid per 100 moles of protein. It appears, therefore, that although substrate is tightly bound to the enzyme, not enough is present to account for all the activity in the reaction mixtures where DPGA is excluded. Phosphate-labeled DPGA was isolated from fluoride-poisoned fermentation mixtures by barium p r e ~ i p i t a t i o nand , ~ was purified by paper chromatography with a 2-propanol/NH40H/water (70.10 20) solvent system. To a solution containing 50 ynioles of DPGX and 200 pmoles of imidazole-HC1 buffer pH 7.0 was added 0.2 ptnole of crystalline enzyme (final volume 5.6 ml,). After incubation and dialysis similar to that described previously, the protein solution was evaporated to dryness which was shown to be I t contained bound P32, linked to the protein: Partial hydrolysis of the protein was carried out by dissolving it in 11 1Y hydrochloric acid and incubating a t 40" for i 2 hours. The hydrolysate, after removal of thc hydrochloric acid, mas passed through a. Domev 50 (H+) resin column '1s described I n I'lnvin ' The eluate was rechromatographed mi piper with the 2-propanol solvent previously described and the region containing the P32-labeledpeptide material was eluted. This material after hydrolysis a t 110' in a sealed tube with 5 7 S hydrochloric acid for 20 hours was chromatographed in the phenolwater system of Block6 Developnit n t of the chromatogram with ninhydrin indicated that the amino x i d s serine, alanine, and glutamic acid were present &long with P32-inorganicphosphate. Prewitiably i n the peptide material resulting from partial hydrolysis the phosphate i q esterified to the Serine hydroxyl. Determination of the Pd2in the phosph(,rylated peptide fraction purified by chroniatographv in0)J Xlayaudon, A A R e n i o n a n d R'i Czl\in Riochr >7 n~ * , I \ ( 4(1n 23, 342 (1957) ( 3 ) l h e c l 4 3PGA was prepared by Ur 2. Pon and tht a u t h o r gratefully acknnnltdge? t h e gift of tht? yampound (4) C S e u b e r i nd H I uqtig A i r h & z o ~ h ~ m 1 i l l (1442) ( 5 ) M F l a i i n 1 Hid C h r m 210, 771 (1954) ( 6 ) R J Block, Anal Chem 22, 1327 (1950).
Vol. 80
dicated that 94y0 of the protein molecules had beeii phosphorylated. It appears that sufficient phosphate is present on the enzyme to account for the observed activity in the absence of DPGA, and that phosphoglyceric acid mutase has a mechanism of action similar to that of phosphoglu~omutase.~ (7) V. A. S a j j a r a n d hl. E. Pullman, Science, 119, 631 (195.4). E. P. Kennedy and D. E. Koshland, J . Bioi. Chenz., 228, 419 ( l 9 . 5 i ) .
DEPARTMEST OF BIOCHEMISTRY UNIVERSITY O F CALIFORSIA
BERKELEY 4, CALIFORNIA r
