Mitochondrial ATPase in the gills of the shore crabCarcinus maenas

Abstract

Posterior gills (No. 7 and 8) of shore crabs Carcinus maenas were homogenized and fractionated by means of differential and density gradient centrffugation. Employment of marker enzymes Na-K-ATPase and carbonic anhydrase for plasma membranes and cytochrome oxidase for mitochondria showed that these structural elements were separated. Ultramicroscopic investigations of combined fractions confirmed the presence of the respective mitochondrial and vesicular plasma membrane structures. An ATPase which did not depend on the presence of sodium (20 raM) ions in the incubation medium but on the presence of potassium (20 mM) ions only was found in the mitochondrial fractions. The mitochondrial ATPase was tightly bound to cellular particulates and activated approximately threefold by bicarbonate (20 mM) ion~. The activity of this ATPase was nearly completely inhibited by oligomycin (1 ~g m1-1) and greatly inhibited by low levels (5 mM) of thiocyanate and calcium ions, the Ki for Ca 2+ being ca 4 raM. The results obtained confirm literature data on high mitochondrial densities in crab gills and allow the assumption of significant rates of energy metabolism in these organs. Considering its properties the mitochondrial ATPase is clearly distinct from crab gilI Na-K-ATPase and can be measured specifically in samples containing Na-KATPase. Mitochondrial ATPase is therefore considered a suitable and reliable marker enzyme for mitochondria.