STUDIES ON THE HISTOCHEMICAL DIFFERENTIATION OF ENZYMES HYDROLYZING ADENOSINE TRIPHOSPHATE

DAVID G. FREIMAN ¹ and NANCY KAPLAN ¹

¹ Departments of Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts

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.

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