Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Magnesium-independent and magnesium-dependent K('+)-stimulated ATPases were found in corn root microsomes and plasma membranes. The magnesium-dependent activity is probably the enzyme described by Leonard and Hotchkiss (Plant Physiol 58, 331, 1976), but the magnesium-independent activity represents a different ATPase with a greater affinity for ATP;A K('+)ATPase isolated from corn root membranes (Benson and Tipton, Plant Physiol. 62, 165, 1978) hydrolyzes free ATP, with maximal activity at pH 7.5. It is specifically stimulated by cations (K('+) is the most effective); stimulation is enhanced by high ionic strength. The Hill coefficient for KCl stimulation is 2 to 3;The purified ATPase is unstable at low ionic strength. Glycine can substitute for salts in preserving activity. Phospholipids stabilize the enzyme at low ionic strength, and both phospholipids and detergents stimulate enzymatic activity. The ATPase is anionic, aggregates at low ionic strength, and adsorbs reversibly to a variety of gel filtration media;When purified ATPase and soy phosphatide liposomes are dialyzed against low-ionic-strength buffer, they form a stable complex which can be isolated by gel filtration or density gradient centrifugation. On reconstitution the pH optimum of the ATPase activity in 50 mM KCl shifts from 7.4 to 8.0, but the pH optimum in 250 mM KCl remains at 7.5. 250 mM KCl partly resolubilizes the reconstituted ATPase;K('+)ATPase membrane vesicles can also be reconstituted from octyl glucoside extracts of corn root or shoot microsomes by adding phospholipids and dialyzing away the detergent. These extracts contain several ATPases, and both magnesium-dependent and magnesium-independent activities are reconstituted. The activity of the reconstituted extracts is greatest below pH 6. The purified ATPase cannot be reconstituted in good yield by this technique;Reconstituted purified ATPase formed tight vesicles which showed no ATP-driven transport. Reconstituted extracts formed vesicles which usually appeared permeable to H('+) and K('+), and could not maintain a membrane potential. They gave no evidence of transport driven by free ATP.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Daniel William Karl
Karl, Daniel William, "Reconstitution and properties of corn membrane ATPases " (1981). Retrospective Theses and Dissertations. 6921.