Magnesium ion activated ATPase and proton transport in vesicles from the gill of rainbow trout (Salmo gairdnerii)

1.(1) Vesicular membrane preparations were isolated from rainbow trout gill homogenates. Magnesium ion dependent ATPase was enriched in the vesicles, either by selective aggregation with magnesium ions, or by density gradient centrifugation.2.(2) The ATPase had optimal activity at pH 6 or below. It had a Km for ATP of about 0.25 mM.3.(3) Dicyclohexyl-carbodiimide at concentrations of 0.2–0.5 mM inactivated the enzyme at pH 7 and room temperature.4.(4) Density gradient centrifugation enriched vesicles showed ATP dependent uptake of acridine orange, which is indicative of proton uptake into everted vesicles. Potassium ions and valinomycin were required for optimal uptake, and because of the high ATPase activity, an ATP regenerating system was required.Acridine orange uptake took place in the presence of 20 μg oligomycin/ml.5.(5) Freeze fracture electron microscopy of density enriched vesicles showed small globular structures affixed to the membrane fragments, apparently on both the convex and the concave side.6.(6) The organization of the membrane preparations in vesicles with a separate internal volume, was demonstrated with the spin labelled probe 2,2,6,6-tetramethyl-4-piperidinone-1-oxyl and electron paramagnetic resonance measurements in the presence of the line broadening agent, chromium oxalate.7.(7) Fluorescein-isothiocyanate labelled dextran was taken up into the cells of isolated gill filaments, by what is suggested to be a process of endocytosis.