266 A SYMPOSIUM ON RESPIRATORY ENZYMES 



P. W. Wilson, University of Wisconsin: 



It is only recently that most investigators of bacterial metabolism 

 have had the opportunity to choose between cell extracts and intact 

 cells. Because of their small size, bacteria as a group have resisted 

 attempts to destroy their cellular integrity. In the past, if studies on 

 bacterial enzyme systems were to be made, the investigator was in 

 the position of Kipling's thief who took the hot stove because there 

 was nothing else that season— he had to use intact cells. Although this 

 limitation has undoubtedly complicated the investigations and the 

 interpretation of the results, it has not been insurmountable. By use 

 of the so-called "resting cell" technique, great strides have been 

 made toward an understanding of the biochemistry of bacteria. With 

 respect to individual enzyme systems the greatest success has at- 

 tended studies in which isolation of the reaction is made possible 

 through choice of substrate rather than enzyme system, for example, 

 hydrogenase and hydrogenlyase. When less specific substrates are 

 employed, great care must be exercised that the interpretation is 

 not oversimplified by applying information gained in what appears 

 to be an analogous study made with the more purified enzyme prep- 

 arations. 



With certain bacterial enzyme systems the investigator is denied 

 even the use of non-proliferating cells. For example, studies on 

 nitrogen fixation by Azotobacter must usually be made with growing 

 cultures : on the symbiotic system with a very complex association of 

 bacteria and host plant. Despite these technical handicaps, the de- 

 velopment of certain methods (1, 2) during the last decade has 

 enabled investigators to secure what Burk has recently described as 

 "the most intimate information that we possess on the mechanism of 

 fixation, and in particular on the nature of the first crucial step in- 

 volved" (3). 



In recent years special techniques have been developed which 

 allow cell-free extracts containing a variety of enzymes to be pre- 

 pared from bacterial species. While it is gratifying that this first step 

 toward isolation of individual enzymes has been made, the imme- 

 diate practical value of the achievement has not been great. In most 

 cases the net result of the separation has been the verification of a 

 portion of the knowledge previously obtained with the resting cells. 

 Recently we have prepared a cell-free Azotobacter "juice" which 

 contains among other enzymes very powerful preparations of hydro- 

 genase and oxalacetic decarboxylase. Our satisfaction over this ac- 



