CHEMICAL COMPOSITION OF THE MEDIA 431 



ions present (particularly the various carboxylates, the proteins, and 

 nucleotides). Finally, it may be noted that different regions or cells of an 

 organ may possess different ionic contents, as has been well shown for heart 

 (Davies et al., 1952; Kiihns, 1954). It is even possible that within an appar- 

 ently homogeneous cell population there occur variations between the 

 individual ceUs (Webb, 1956; Conway, 1957). 



A further difficulty in duplicating an intracellular environment for an 

 enzyme arises from the fact that the distribution of the ions within the 

 cell is unknown; even when the over-all concentration is known, one can- 

 not be certain that this is the concentration in the enzyme region. In the 

 first place, a fraction of each ion population may be bound. This applies 

 particularly to ions such as Mg"*""^ or Ca"^"^, the actual concentration of the 

 free ions possibly being very low, but also to K"^ and Na"^. Myosins A and 

 B bind K"^ and Na"^ on the imidazole and amino groups (Lewis and Saroff, 

 1957). A total of around fifty ions may be bound per 10^ gm of protein on 

 a molar basis and the dissociation constants for myosin A are 1.25 X 10~^ M 

 for K"^ and 6.25 X 10"^ M for Na"^ with respect to the imidazole sites. Thus 

 appreciable amounts of these ions may be removed from solution in muscle 

 cells. In the second place, there is evidence that mitochondria can possess 

 different ionic concentrations than the external medium (Macfarlane and 

 Spencer, 1953; Davies, 195-4; Ulrich, 1960). Apparently both K+ and Na+ 

 may be at higher concentrations within the mitochondria, although how 

 much of this is due to binding is not known. Analysis of nuclear, mito- 

 chondrial, microsomal, and supernate fractions of liver cells for K"^, Na"*", 

 Ca"^"^, and Mg"^"^ has shown definite differential distribution (Griswold and 

 Pace, 1956). While K"^ and Na"^ are mainly in the supernate (53% and 66% 

 respectively), Ca '""^ is highest in the mitochondria, and Mg"^"*" is highest in 

 the microsomes. Studies of the exchangeability of intracellular ions and the 

 rates of efflux of radioactive ions from cells have usually indicated different 

 compartments, fractions of the total ion content having different mobilities. 

 In erythrocytes the K"^ is divided between at least two compartments 

 (Solomon and Gold, 1955) while in muscle the Na"^ is not homogeneously 

 distributed (Conway, 1954). Similar results have been obtained with these 

 and other ions on a variety of tissues. Summarizing this evidence, it must 

 be admitted that the concentration of an ion in the enzyme region may 

 be either higher or lower than indicated by the over-all concentration. 



The effects of the organic compounds of the cell on enzyme properties 

 are mostly obscure. Certain substances, such as glutathione and ascorbic 

 acid, may regulate activity through redox equilibria with the enzyme 

 SH-groups, or they may exert a protective action against inhibition, 

 and their absence in reaction media may modify the enzyme appreciably. 

 On the other hand, the use of certain substances in media to increase the 

 activity or to stabilize enzyme preparations may create abnormal con- 



