VOL. 12 (1953) ENZYMIC CONVERSION OF PHOSPHORYLASE 237 



the sedimentation patterns of reaction mixtures in which increasing amounts of phos- 

 phorylase a have been converted to the b form. The purified PR enzyme used in these 

 experiments represented no more than three % of the total protein present, and the 

 concentration did not exceed 0.015%. At this concentration no protein peak for the 

 PR enzyme would be visible. A second protein component, with a sedimentation con- 

 stant of 8.2, can be seen to arise at the expense of phosphorylase a. In Fig. lE the cjn- 

 version is almost complete. The area of the smaller protein in % of the total protein is 

 at all times equivalent to the enzymically determined proportion of phosphorylase h. 

 Addition of adenylic acid (o.ooi M) to the enzyme solution during ultracentrifugation 

 had no effect on the rate of sedimentation nor on the relative proportions of the two 

 proteins. 



The protein product of the PR-catalyzed reaction behaves as a single molecular 

 species throughout the course of centrifugation. This suggested that phosphorylase a 

 is split b}^ PR into equal or nearly equal parts. Molecular weight determinations have 

 shown this to be the case. The molecular weights of phosphorylases a and b have been 

 calculated using the constants and formula listed in Table L The partial specific 

 volumes ( F20) were determined, using the Linderstrom-Lang gradient tube as described 

 by Taylor*. 



TABLE I 



MOLECULAR CONSTANTS OF PHOSPHORYLASES a AND b 



Constant Phosphorylase a. Phosphorylase b. 



* * 



S20, a,* 13-2 8.2 



i^jo, i4,Xlo^ 2.6 3.3 



F20 ^^^ 0-751 0.751 



M.W. *** 495,000 242,000 



* expressed as Svedberg units 

 ** determined by Green^ pj- 



*** calculated from M.W. = ^ 



The sedimentation constant of phosphorylase a reported here, namely 13.2, is of the 

 same order as that found by Oncley in 1943 which was 13.7. Phosphorylase b was not 

 centrifuged at that time. 



The diffusion constants for enzymes a and b call for some comment. Oncley* 

 determined the diffusion constant for phosphorylase a in a neutralized cysteine-glycero- 

 phosphate buffer. The experiment was done at 25° C. Pictures were taken between 625 

 and 2036 minutes. Oncley reports formation of insoluble cystine as well as the appear- 

 ance of skewed curves during the course of the experiment. Despite these difficulties a 

 value between 3.2* and 3.8* seemed reasonable. The molecular weight calculated by 

 Oncley was set between 340,000 and 400,000. 



Green, in 1944, determined the diffusion constant of phosphorylase b and found 

 it to be 3.3''*. The apparent similarity of diffusion constants for phosphorylase a and b 

 pointed to molecular weights of the same order of magnitude. 



In the present experiment, versene (ethylenediaminetetraacetic acid) was used 

 instead of cysteine to solubilize phosphorylase a, and a five-time recrystallized prepara- 

 tion of enzyme was used. The experiment was run at 2° C. The same value (2.6) was found 



* ^iO.w X lO^ 



References p. 238. 



