The localization of (Na+-K+) ATPase in the intact electrocyte of the electric organ of Electrophorus electricus (L.) and its subcellular fractions was investigated by biochemical and cytochemical methods. The distribution of AChE activity in the subcellular fractions was also comparatively analysed with this enzyme serving as a marker of the innervated membranes of the electrocyte. After application of cytochemical method of Farquhar and Palade to glutaraldehyde-fixed tissue, reaction was observed only at the membranes of vesicles localized at the periphery of the electrocyte. Previously fixed electrocytes, incubated in Ernst's medium showed reaction only at the vesicles whereas in unfixed tissue reaction also appeared at other membranes (surface and invaginations) of the anterior and posterior faces. This reaction was significantly inhibited in the presence of ouabain or in the absence of K+. Inhibition of Na+-K+-ATPase by glutaraldehyde fixation was also confirmed by biochemical analysis.
Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freeze-fracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.
Electrocyte membranes of Electrophorus electricus exhibit high ATPase activity, as demonstrated by cytochemical and biochemical techniques. This activity is visualized as electron-dense deposits in electron micrographs, and appears to be localized only at the innervated face of the electrocyte. ATP hydrolysis can be detected cytochemically or biochemically only in the presence of calcium or magnesium. The effects of Ca or Mg on ATPase activity can be described by Michaelis-like functions with similar apparent Km values for Ca and Mg (0.41 mM and 0.23 mM, respectively). Vmax, however, is fivefold higher in the presence of Mg. The effects of the two cations are not additive, and pH dependence of ATP hydrolysis is identical in the presence of Ca or Mg (maximal at pH 8-9). Therefore, it can be concluded that Ca and Mg activate the same enzyme, the differences in Vmax being attributable to influences in kcat.
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