FURTHER STUDIES OF THE APPARENT ADENOSINETRIPHOSPHATASE ACTIVITY OF CELL MEMBRANES IN FORMOL-CALCIUM-FIXED TISSUES
Authors: NOVIKOFF ALEX B. , DRUCKER JOAN , SHIN WOO-YUNG , GOLDFISCHER SIDNEY 
Published: April 2011

1. The Wachstein-Meisel and Padykula-Herman media for adenosinetriphosphatase activity appear to demonstrate the same enzyme activities in cell membranes. Calcium ions can replace magnesium ions in the Wachstein-Meisel medium without diminution of staining intensities in the membranes. Indirect evidence suggests that the enzymes demonstrated by the staining methods are calcium-activated when tested biochemically 2. No support is found for the view that the lead ions present in the medium make the Wachstein-Meisel method incapable of demonstrating mitochondrial adenosinetriphosphatase. Formol-calcium fixation, not the lead ions, inhibits mitochondrial ATPase under the conditions employed. 3. Variation in pH (5.5-7.9), and the presence or absence of sodium ions and ouabain have no demonstrable effect upon cell membrane staining for apparent adenosinetriphosphatase activity in frozen sections of formol-calcium-fixed tissue. This, like the absence of differential effects of calcium and magnesium ions, may reflect properties of the enzymes or of the milieu in tissue sections. 4. The specificities of the membrane phosphatases are examined. Specific adenosinetriphosphatase (or nucleoside phosphatase) appears to be in the cell membranes delimiting the lateral borders of the cells of uterine epithelium of the rat; the interdigitations of adjacent cells in the distal convolutions, thick limbs of Henle, and collecting ducts of the rat kidney; the Purkinje cells and other neurones in the rat cerebellum; the brush borders of the cortical parts of the proximal convolutions; and perhaps the egg and follicle cell extensions in some Graafian follicles of the mouse. Less specific nucleosidephosphatases such as apyrase, or a combination of specific triphosphatase with enzymes like ADPase, appear to be in the membranes at the luminal surface of uterine epithelium in the rat and the bile canalicular and sinusoidal surfaces of the parenchymatous cells of rat liver, in the membranes of rat hepatoma, endothelial cells and smooth muscle of arterioles, neuroglia and neuropil of rat cerebellum, capillaries of rat kidney, hepatoma and brain, and fibroblasts and venules in rat uterus, and in the zona pellucida of most Graafian follicles in the mouse ovary. 5'-Nucleotidase activity appears to be present in the cell membranes at the sinusoidal and canalicular aspects of hepatic cells of the rat, between adjacent cells of the germinal epithelium in the mouse ovary, of the capillary endothelium of tumors and other tissues and of some fibroblasts in the rat uterus. Alkaline phosphatase activity is apparently present in the membranes of the endothelium of rat brain and uterine capillaries, some areas of luminal surfaces and bile canaliculi in the rat, and some tumor cells, as well as the brush borders of the proximal convolutions of the kidney. 5. It is not known if the various phosphatases perform the same or different functions in different cells. Attention is drawn to the special interest of the observations described for the ovary and uterus. 6. In formol-calcium-fixed frozen sections of mouse oviduct and rat uterus the myofibrils of the smooth muscle cells dephosphorylate adeno-sine-tri-, -di-, and -5'-phosphates. Staining is most pronounced with the triphosphate and least pronounced with the mono-phosphate as substrate. The inner and outer muscle layers of rat uterus are equally stained with adenosine monophosphate as substrate, whereas with the di- or tri-phosphate the outer layer is stained more intensely. 7. Holt's procedure of storing fixed tissues in cold gum acacia-sucrose and Feder's method of embedding in polyvinyl alcohol may help preserve for long periods both cell structure and enzyme activities. The eznymes include, in addition to the nucleotide phosphatases, the less specific alkaline and acid phosphatases, non-specific esterase, and di- and tri-phosphopyridine nucleotide-tetrazolium (nitro-blue tetrazolium) reductases.