584 PETER MITCHELL 



and sugars into the metabolic systems of Escherichia coli and Pseudomonas 

 sp. resemble enzyme systems in being inducible, and that the induction 

 can be blocked by certain inhibitors of protein synthesis. These kinetic 

 observations lend further support to the idea that the transport catalysts 

 may be normal enzyme and catalytic carrier systems. But we must be 

 careful not to imagine, as some microbiologists have done, that kinetic and 

 inhibitor studies of the behaviour of whole cells or protoplasts can reveal 

 the composition of the catalysts immediately involved in the transport 

 processes — for example, whether they are proteins or not. The only 

 unequivocal way of characterizing the catalysts of transport is to isolate 

 and purify them, and to examine their structure and function by direct 

 analytical and kinetic methods. 



Let us now turn to the third method of attack on the membrane 

 transport problem — the study of the composition of the plasma membrane. 

 This phase of the work has its origins in the isolation of a small-particle 

 fraction from disintegrated micrococci which Dr. Moyle and I found to be 

 a lipoprotein, just sufficient in amount to have originated by the frag- 

 mentation of the plasma membrane, and containing an acid phosphatase 

 which, in intact cells, we knew to be accessible to glycerophosphate from 

 outside [27, 18]. I should, perhaps, say at this point that the problem of 

 isolating the plasma membrane material from bacteria is made com- 

 paratively easy by the very small size of the cells and their large ratio of 

 area to volume, a membrane only 10 m^ thick at the surface of the proto- 

 plast representing 5-10*;' ,, of the dry weight of the cell. 



The isolation of the membrane material in a morphologically recog- 

 nizable state owes much to Dr. Weibull's discovery that the cell wall of 

 certain bacteria could be removed enzymically without breaking the 

 plasma membrane as long as the protoplast was prevented from swelling 

 by the addition of sucrose or other osmotically effective solute to the 

 suspension medium [28]. When the suspension medium was suddenly 

 diluted after removing the cell walls from the protoplasts, the contents of 

 the protoplasts were thrown out, and the membranes, looking like little 

 bursted balloons in light and electron microscopy, could be collected on 

 the centrifuge [29]. In this way. Dr. Weibull was able to show that the 

 cytochrome pigments of Bacillus megaterium sedimented in the membrane 

 fraction ; but as this organism unfortunately contains very many cytoplas- 

 mic particles which also sedimented with the membrane fraction, it was 

 not certain whether the cytochrome pigments belonged to the membrane 

 or to the adhering particles. Dr. Moyle and I therefore undertook a 

 similar type of fractionation on staphylococci, which are comparatively 

 free of cytoplasmic particles [30]. We relied upon a controlled autolytic 

 method to weaken the cell wall before diluting the cell suspension to burst 

 the protoplasts and liberate morphologically recognizable membranes. It 



