At least 3 enzymes or groups of enzymes present in dog kidney and jejunum are capable of splitting adenosine triphosphate at pH 9.4. In addition to nonspecific alkaline phosphatase and what appears to be a true adenosine triphosphatase, an enzyme behaving like a less specific organic polyphosphatase is present in small blood vessels and smooth muscle. These enzymes can be readily differentiated by various inhibitors such as cysteine, ethylenediamine tetraacetic acid, 80% ethanol, pretreatment in acetate buffer at pH 4.0 with or without added chloride or nitrate ion, or by incubation at 4°C. Differential effects can also be obtained by pretreatment with lead or zinc ion at pH 6.0. Of the substrates tested only inosine triphosphate gives results comparable with adenosine triphosphate. All other substrates, including various nucleotides and thiamine pyrophosphate, produce patterns similar to that obtained with adenosine diphosphate, or with adenosine triphosphate after pretreatment with buffer at pH 4.0. These observations suggest that an enzyme less specific than adenosine triphosphatase is being demonstrated. The similar patterns obtained following incubation in lead-adenosine triphosphate substrate at pH 7.2 also indicate that true adenosine triphosphatase cannot be shown by this method. 5'-Nucleotidase plays little role in these reactions under the experimental conditions used. Enzymes behaving in similar fashion have been noted in some human tissues. Of particular interest is a broad inner zone of the circular muscle coat in the small intestine of the dog which is strongly positive for adenosine triphosphatase and polyphosphatase, but negative for 5'-nucleotidase. A similar, but much narrower zone has been noted in the ileum and colon of man.

Pretreatment of cryostat sections of dog kidney with sodium salts of various anions in acetate buffer at pH 4.0 for periods of time ranging up to 6 hours produces differential inhibition of non-specific alkaline phosphatase activity as determined by the Gomori method. The degree of anionic effect is dependent on the nature of the anion and modified by the cation present. The effect also varies directly with the anionic concentration, duration of exposure and temperature of the pretreatment buffer and inversely with the pH. On the basis of the differential suppression of enzymatic activity by the various anions, and the conditions under which these effects were evident, distinctive inhibition profiles were established for the several phosphomonoesterases tested, including the renal and intestinal alkaline phosphatases and the smooth muscle 5'-nucleotidases of several species. The similarity of the results obtained in various biochemical studies on other enzymes reported in the literature and those obtained histochemically in these experiments suggests a common mechanism of action; this is presumed to be a differential effect of anions on the dissociation of apoenzyme and coenzyme. The method described provides a simple and effective means of differentiating, under histochemical conditions, closely related and even apparently identical enzymes.