THE SPECIFICITY OF THE HISTOCHEMICAL METHOD FOR ADENOSINE TRIPHOSPHATAS

Three categories of phosphatase activity toward adenosine triphosphate at pH 9.4 have been isolated histochemically: (1) phosphatase which requires -SH groups; (2) phosphatase which is inhibited by -SH groups; and (3) phosphatase which is relatively indifferent to -SH groups. This enzymatic activity was demonstrated in thin (5 µ), unfixed, frozen sections. True adenosine triphosphatase has been separated histochemically by virtue of its -SH dependence. It was demonstrated in cardiac and skeletal muscle fibers and in the mitochondria of the kidney tubules. The staining of these structures can be prevented by treatment with an -SH inhibitor, such as p-chloromercuribenzoate or salyrganic acid. Also their stainability can be restored by reversing the inhibition of the enzymes with -SH compounds, such as BAL or cysteine. There was no reaction of these particular structures when adenosine diphosphate or adenosine-5-phosphate was used, suggesting that under the conditions of this histochemical test only the terminal phosphate of adenosine triphosphate is removed. Phosphatase activity of the smooth muscle of the intestinal muscularis toward adenosine triphosphate differed from that of striated muscle in that it was less sensitive to p-chloromercuribenzoate. Sulfhydryl compounds, such as BAL or cysteine, enhanced the staining of the above mentioned sites of true adenosine triphosphatase activity. On the other hand, these -SH compounds are powerful inhibitors of alkaline phosphatase activity, especially toward adenosine-5-phosphate. When adenosine triphosphate was the substrate, BAL prevented the staining of the brush borders of the kidney tubules and intestinal epithelium, thus suggesting the participation of alkaline phosphatase in the dephosphorylation of adenosine triphosphate. Furthermore, the staining of these brush borders was unaffected by the -SH inhibitors. The strong phosphatase activity of endothelium and vascular smooth muscle toward adenosine triphosphate was seemingly indifferent to -SH groups, since the staining of these structures was not markedly influenced by -SH inhibitors or compounds. The nature of this staining has not been elucidated. Comparisons of the localization of phosphatase activity toward adenosine triphosphate, -diphosphate or -5-phosphate were made in the ventricle, tongue, kidney, and duodenum. Differences in the localization of phosphatase activity toward adenosine triphosphate and adenosine-5-phosphate were also made in liver, lung, spleen, uterus, and aorta.