Peptides with different chromatographic and electrophoretic properties were obtained from human placental and renal alkaline phosphatases by tryptic digestion of the enzymes labelled with radioactive orthophosphate at their active centres. These results provide structural evidence for the distinct genetic origins of the two isoenzymes that had previously been inferred from their different properties and from the observed phenotypic variation of placental phosphatase.

The formation of 1,25-dihydroxycholecalciferol from 25-hydroxycholecalciferol by chick kidney homogenates is inhibited by increasing concentrations of Ca 2+ . The apparent K m for the hydroxylation reaction is 1×10 -7 m , significantly lower than that reported for isolated mitochondria. Separated cytoplasmic and particulate fractions are inactive, but on recombination activity is restored, possibly because of the presence in the soluble fraction of a factor with a high affinity for 25-hydroxycholecalciferol.

Subcutaneous injection of isoprenaline into rats produces a rapid rise in alkaline phosphatase activity which is largely confined to the right atrium of the heart. The phosphatase activity of extracts of atrial tissue from treated animals is on average four times that of controls. The rise in activity is largely suppressed by previously treating the animals with cycloheximide or actinomycin D. Comparison of the properties of the alkaline phosphatase in extracts of atria from isoprenaline-treated rats with control preparations shows the stimulated enzyme activity to be similar to that present in the resting state. In a number of respects, including substrate specificity, response to activators and inhibitors, rate of loss of activity on heating and electrophoretic mobility of the native and modified enzyme, the atrial phosphatase resembles alkaline phosphatases from other mammalian non-intestinal and non-placental tissues. These results suggest that the effect of isoprenaline is to increase the rate of synthesis of atrial alkaline phosphatase, and the ability of the tissues of the right atrium to respond in this way may be related to the rich sympathetic innervation of this part of the heart.

1. The differential effects of adding Zn 2+ and Mg 2+ on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn 2+ , inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn 2+ –pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg 2+ also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg 2+ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.

1. Inhibition of the pyrophosphatase and orthophosphatase activities of human liver and small-intestinal alkaline-phosphatase preparations by different classes of inhibitors has been studied. 2. Each type of substrate, pyrophosphate or orthophosphate, is a competitive inhibitor of hydrolysis of the other type. 3. l-Phenylalanine is a non-competitive inhibitor of both types of activity of the intestinal preparation, but inhibits neither activity of the liver enzyme. Arsenate is a competitive inhibitor of both activities of both preparations. For a given inhibitor, the values of K(i) are independent of the type of substrate used when measurements are made at the same pH. 4. Mg(2+) ions activate orthophosphatase but inhibit pyrophosphatase, except in very low concentrations. 5. These results are compatible with the presence in each tissue preparation of a single enzyme with one type of active centre, possessing both orthophosphatase and pyrophosphatase activities.