A B S X R A C X We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25"C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO~ a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3-is established along with a pCO~ gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-ram-long cellulose particles, no facilitation of CO~ diffusion was detectable. A mechanism of facilitated CO~ diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxes predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism, CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold.

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An assay of adenosine(5′)tetraphospho(5′)adenosine (Ap4A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap4A in cellular extracts. In eukaryotic cells this method yielded levels of Ap4A varying from 0.01 μM to 13 μM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G1‐arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap4A pools gradually increased 1000‐fold during progression through the G1 phase reaching maximum Ap4A concentrations of about 10 μM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 μM) in a “committed” but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap4A pool. Cells lacking a defined G1 phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap4A (about 0.3 μM) even during mitosis. From this high level, Ap4A concentration increases only about tenfold during the S phase. Temperature‐down‐shift experiments, using chick embryo cells infected with transformation‐defective temperature‐sensitive viral mutants(td‐ts), have shown that the expression of the transformed state at 35°C is accompanied by a tenfold increase of the cellular Ap4A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap4A levels within 20 h. The possibility of Ap4A being a “second messenger” of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap4A is a ligand of mammalian DNA polymerase α, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.

The steady-state CO2 flux across thin layers of 30 g/100 ml albumin solutions was measured in two different C02 partial pressure ranges (boundary Pco 2 values 3 and 8 torr, and 160 and 650 torr, respectively). From the data the apparent diffusion coefficient for CO2, Dco 2 , was calculated. In the high Pco 2 range a value of Dco 2 was found which is to be expected on the basis of diffusion of dissolved CO2 only. In the low Pco 2 range Dco 2 was about 100 % higher than in the high Pco 2 range, when carbonic anhydrase was present and the pH was -7.7. Dco 2 depended on the concentration of carbonic anhydrase. It increased with increasing pH. It is concluded that an additional diffusion of bound CO 2 (facilitated CO2 diffusion) occurs in the low Pco 2 range and that this transport involves the hydration of CO2. From the diffusion coefficients in the two Pco 2 ranges the rate of facilitated diffusion was determined. Approximate calculations show that this rate (at pH < 7.7) can be explained on the basis of the proposed mechanism of facilitated CO 2 diffusion: bicarbonate diffusion and simultaneous proton transport by albumin diffusion. The view that facilitated CO 2 diffusion is mediated by the diffusion of albumin is supported by the experimental finding of a considerable suppression of the facilitated CO2 flux in the presence of gelatinized agar-agar.

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