ENZYME-SUBSTRATE COMPOUNDS 



struction of the glycolytic system with purified enzymes. But 

 such a reconstruction may be fauky with respect to the relative 

 amounts of enzymes and coenzymes that are added, the dif- 

 fusibility of the coenzymes, and, in addition, the steady-state 

 concentrations of the intermediates in the system, for the in- 

 tracellular system shows complex reaction patterns (20). 



Reconstruction of the respiratory chain appears to be a 

 much more formidable problem. Cytochrome c, the only mem- 

 ber of the chain that has been purified, has an activity in the 

 reconstructed Keilin and Hartree system of less than 1% that 

 of the intact system (61), although in recent experiments with 

 the succinic oxidase liver system this value has been raised 

 towards 50% (R. Estabrook, unpublished data). And such 

 other anomalies beset the reconstruction as the fact that isolated 

 cytochrome c will readily bind cyanide, whereas cytochrome c 

 in the intact respiratory chain does not (69). 



Separation of some parts of the oxidase system has not yet 

 been accomplished; although the cytochromes a^ and a have 

 been purified in the presence of detergents, no chemical separa- 

 tion of the components has been obtained (64). The degree of 

 purification of this portion of the respiratory chain obtainable 

 when phosphorylative activity is retained, appears to be even 

 less, although a smaller phosphorylating particle has been 

 obtained by digitonin treatment (46a). 



In order to evaluate any of these reconstructions it is es- 

 sential to compare them directly with quantitative measurements 

 of the intact enzyme systems, especially in the case of the re- 

 spiratory chain involved in oxidative phosphorylation. Spec- 

 troscopic methods can be used to detect six intermediates in 

 this respiratory chain, cytochromes As, a, c, and b, flavoprotein, 

 and pyridine nucleotide (26,28). Spectroscopic identification 

 plus kinetic and inhibitor studies can be used to: (7) establish 

 the sequence of action of the components of the chain; (2) 

 identify components not detectable spectroscopically if they 

 {a) react specifically with a substrate or inhibitor or {b) can be 

 made to be rate-limiting. 



319 



