1H/2H isotope exchange studies in intact erythrocytes.

ethanol. Kinetic measurements on treated enzyme that had been separated from the modifying agent by gel filtration showed that K, values were unaffected and that inactivation was purely a V,,,. phenomenon. Thus chemical modification leads in both of the cases studied to totally inactive enzyme. The action of phenylglyoxal is presumably on an essential arginine residue@), which may be at, or close to, the active site, since the competitive inhibitor P, provides protection. The behaviour of the intestinal enzyme resembles that of pig kidney and E. coli alkaline phosphatases. This similarity is expected for an active-site residue, since it would belong to a class of highly conserved residues in alkaline phosphatase proteins. The non-protection by PI of attack by iodoacetamide may be indicative of a more remote location for the particular residues involved. Iodoacetamide sensitivity is not shared by the kidney enzyme (M. Wass & P. J. Butterworth, unpublished work), which again may argue for the sensitive residue being remote from the active centre. We cannot yet be sure of the nature of the group attacked by iodoacetamide. The effect of pH on the reaction and the ineffectiveness of N-ethylmaleimide point to some other group than thiol. Further investigations are needed to answer this question.