EFFECTS OF BUFFERS 839 



phosphor ylase, this enzyme being inhibited 41-78% by 70 vaM Na+. K+ 

 stimulates this transphosphorylase and part of the activation could result 

 from this. The actions of inhibitors on this system would certainly be al- 

 tered by the concentrations of these ions. Such results indicate how impor- 

 tant it is to perform inhibition experiments in as physiologically normal 

 media as possible if conclusions as to the effects in living cells are to be 

 drawn. Effects other than directly on the enzyme systems may also be 

 important. The stimulation of anaerobic glycolysis in slices of guinea pig 

 brain by Ca++ was found to be due to actions on the cell membrane (Adams 

 and Quastel, 1956). Mitochondria are also sensitive to salts. The oxidations 

 of acetate and the tricarboxylic acid cycle intermediates are stabilized by 

 K+ but not by Na+ in heart mitochondria; indeed, Na+ is usually inhibitory 

 (Korff et al., 1954). The soluble acetate-activating enzyme is stimulated 

 by K+ but it is not known if this is involved in the stabilization. It may 

 well be that the effects of these ions are on the structure of the mitochon- 

 dria and it is important in all work with particulate fractions to bear in 

 mind the possibility of such structural changes. 



EFFECTS OF BUFFERS 



Most enzyme and metabolic reactions are studied in media containing 

 buffers of various types. Buffer ions may specifically stimulate or depress 

 enzymes; may interfere or react with substrates, inhibitors, or cofactors; or 

 may exert ionic strength effects. The effects of buffers have been mentioned 

 several times in various connections and the quantitative formulation by 

 Alberty was presented in Chapter 14. Nevertheless, the ubiquity and impor- 

 tance of this problem warrants further discussion and illustration. It is 

 well to emphasize that the buffer concentration should be as low as possible, 

 commensurate with maintenance of a satisfactory pH, and that the media 

 should be made more physiological and the ionic strength normalized by 

 the addition of KCl or other inorganic or organic ions. It might also be well, 

 wherever possible, first to determine the activity of the enzyme in neutral 

 salt solutions of the appropriate ionic strength and then to determine the 

 effects of various buffers, keeping the ionic strength constant, so that the 

 buffer with the least effect on the enzyme may be chosen. 



Examples of inhibitions of enzymes by anions have been known for 50 

 years and in some cases these are the anions of buffers commonly used. For 

 example, phosphate has been shown to inhibit carboxypeptidase (Smith 

 and Hanson, 1949; Neurath and de Maria, 1950), fumarase (Massey and 

 Alberty, 1954), urease (Kistiakowsky et al., 1952), phosphoglucomutase 

 (Klenow, 1955), carboxylase (Stewart, 1957), arylsulfatase (Webb and 

 Morrow, 1959), and other enzymes. The inhibition of plant cytochrome 

 oxidase by sodium bicarbonate (Fig. 15-22) is partly specific and partly 

 an ionic strength effect (Miller and Evans, 1956). The effect of the bicarbo- 



