APPROACHES TO THE ANALYSIS OF SPECIFIC MEMBRANE TRANSPORT 589 



(strain ML 30) with these assumptions in mind. We found, as illustrated 

 in Tables II and III, that after disintegrating washed suspensions of 

 Escherichia coli mechanically, and fractionating on the centrifuge, some 

 85% of the glucose-6-phosphatase was present in the clear solution that 

 had been centrifuged at 35 000 g for i hr., and some i2"o was initially 

 present in the cell envelope fraction which centrifuged down at 2000 g in 

 30 min. On re-submitting the cell envelope fraction to the disintegration 

 procedure, to release any enzyme that might have been trapped by re- 

 closure of some of the membranes, a further 5"o of the enzyme was ob- 

 tained in the " soluble " form, bringing the amount of " soluble " glucose 6- 

 phosphatase recovered to some 90" ^ of the whole. These and other related 



TABLE III 



Distribution of Glucose 6-phosphatase in Esrhen'cliia cnli (ML 30) 



Glucose 6-phosphatase ,, „ , 

 T- . ^ . ^ "o 1 otal 



r raction activity . . 



(/umole P/g. min.) 



From ivhole cells 



"Soluble", Si i-8o 84-7 



Very small particles, P3 0064 3-0 



Cell envelopes. Pi 0263 12 3 



From redisintegrated cell envelopes 



"Soluble", S2 0-098 4-6 



Cell envelopes, P4 0-128 6-0 



Intact untreated cells z- 11 99 



experiments showed that although the glucose-6-phosphatase probably has 

 an affinity for a cell envelope component, according to the usual standards 

 it would be classed as a soluble enzyme. We discovered, however, as 

 illustrated at the bottom of Table III, that the rate of hydrolysis of 

 externally added glucose-6-phosphate by suspensions of intact cells 

 represents the full expression of the "soluble" enzyme activity, and, as 

 shown in Table IV, the activity of the intact cells was little affected by 

 breaking the plasma membrane with benzene (5*^0 v. /v.) or by freezing 

 and thawing. We showed that glucose-6-phosphate does not penetrate into 

 the protoplast of intact cells, for although it could be fermented rapidly by 

 cells in which the membrane was ruptured, in intact cells it w'as fermented 

 only at a rate corresponding to that of the liberation of free glucose by the 

 fully expressed glucose-6-phosphatase. Further, the glucose-6-phosphatase 

 of intact cell suspensions was found to liberate the inorganic phosphate of 

 externally added glucose-6-phosphate, not in the protoplasts, but in the 



