PHOSPHORUS METABOLISM 99 



acetate is added. For example, if Ehrlich ascites cells are incubated in ni- 

 trogen for 1 hr at 37° the P,; level is high; when glucose is added there is a 

 rapid decrease in P, (Laws and Stickland, 1962). lodoacetate at 1 niM 

 completely prevents this fall in P, anaerobically; no hexose phosphates can 

 be formed because there is no ATP. Aerobically the inhibition is 75%, in- 

 dicating other pathways for phosphorylation. When ATP is initially high, 

 or is added, P, will decrease in the presence of iodoacetate. 



The uptake of phosphate by cells may not be entirely a simple inward 

 diffusion to replace intracellular phosphate incorporated metabolically. A 

 very interesting study of phosphate intake by the yeast cell was made by 

 Leggett (1961) on the basis of a carrier mechanism in the membrane. The 

 uptake is coupled with the synthesis of ATP associated mainly with the 

 3-PGDH reaction; one component is rate-limited by the phosphorylation of 

 ADP and a second by the hexokinase reaction. A third minor component 

 is associated with the oxidation of cytochrome b. Inasmuch as the phos- 

 phate uptake is related predominantly to 3-PGDH activity, it is not sur- 

 prising that iodoacetate readily inhibits it. Leggett claims that iodoacetate 

 reduces the concentration of a carrier-P complex and inhibits phosphate 

 uptake uncompetitively (since the maximal uptake rate is changed and K,,^ 

 is not, the inhibition must be classed formally as noncompetitive, although 

 what this means kinetically it is difficult to say). Unfortunately, Leggett 

 used 10 mM iodoacetate so that any interpretation of the results based on 

 a selective glycolytic action must be accepted cautiously. 



The most important aspect of phosphorus metabolism for our purpose is 

 the levels of adenine nucleotides and creatine-P within cells and the effects 

 of iodoacetate upon them. Synthesis of ATP should be depressed aerobically 

 and to a degree dependent on the activity of iodoacetate-resistant pathways 

 for oxidative phosphorylation. Any uncoupling action exerted by iodoace- 

 tate (see page 61) will also be of importance. Early workers showed de- 

 creases in the creatine-P of nerves and muscle (Gerard and Tupikow, 1931) 

 and the heart (Burns and Cruickshank, 1937) following treatment with iodo- 

 acetate, which is probably a consequence of the fall in ATP. Decreases in 

 cellular ATP levels have been reported in Aspergillus niger (Mann, 1944), 

 human erythrocytes (Prankerd and Altman, 1954), intestinal mucosa (Feher 

 et al, 1956), calf lens (Nordmann et al, 1954), guinea pig seminal vesicle 

 mucosa (Whittam and Breuer, 1959), and Ehrlich ascites cells (Thomson 

 et al, 1960). In most cases rather marked effects have been observed at con- 

 centrations of iodoacetate around 1 mM. In some instances the fall in ATP 

 is accelerated by its utilization, as in the formation of hexose phosphates 

 in brain extracts (see tabulation on page 75) (Geiger, 1940), or by cell 

 activity, or by ATPases. The effect of any inhibitor on ATP level will depend 

 on the balance of synthesis and breakdown. lodoacetate produces primarily 

 an inhibition of synthesis, as shown by Nakao et al. (1960) in erythrocytes, 



