APPROACHES TO THE ANALYSIS OF SPECIFIC MEMBRANE TRANSPORT 593 



that the membrane contains a fixed number of substratum sites at which 

 this soluble enzyme can be specifically bonded. We designed a series of 

 experiments to decide between these alternatives, and I can best indicate 

 briefly our conclusion by the example from part of an experiment shown 

 in Table IV. The method of approach was to inactivate the membrane- 

 located enzyme in the intact cell with an irreversible inhibitor (in this case, 

 iodoacetate at pH 8), and then to determine whether the enzyme could be 

 replaced in vitro by the "soluble" enzyme of the normal protoplasm 



TABLE V 



EQUn.IBRATION OF a-KETOGLUTARATE DEHYDROGENASE BETWEEN THE 



Membrane and Protoplasm of Micrococcus lysodeikticus 



Membrane "Protoplasm" 



Normal cells 55-5 44-5 



lodoacetate-treated cells ^ 14-6 4-5 



lodoacetate-treated membrane (i) 14-6 — 



Normal " Protoplasm" (2) — 40 o 



After re-separating mixture of (i) and (2) 35 '5 20 'O 



fraction. The numbers in Table \" represent a-ketoglutarate dehydro- 

 genase activity expressed as a percentage of the total activity of the normal 

 intact cells, which, in this experiment, had an absolute value of 0-65 /^mole 

 substrate per g. cell dry weight per minute. Rather more than half the 

 total activity was initially present in the membrane fraction in this batch 

 of cells. After reacting the intact cells with iodoacetate and separating the 

 membrane and "protoplasm" fractions as usual, the total a-ketoglutarate 

 dehydrogenase activity was reduced to about 20" ,3 of the normal. A sample 

 of the inactivated membrane fraction, representing an activity of 14-6, was 

 now thoroughly mixed with a sample of the normal " protoplasm" fraction, 

 representing an activity of 40, and the two fractions were separated again 

 on the centrifuge. As shown in Table V, the activity of the membrane 

 fraction was found to have risen by 20-9 units, while that of the "proto- 

 plasm" had fallen by 20-0 units — an equivalent amount within experi- 

 mental error — showing a substantial transfer of enzyme from the " soluble " 

 state in the "protoplasm" fraction to the bound state in the membrane 

 complex. This, and other confirmatory and related experiments imply that 

 the distribution of a-ketoglutarate dehydrogenase activity in Micrococcus 

 lysodeikticus does indeed depend upon the mutual satisfaction of locational 

 affinities between a soluble a-ketoglutarate dehydrogenase and a specific 

 substratum in the plasma membrane complex of the cell, 

 vol.. n. — 2 Q 



