Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet.

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg(2+) and K(+). The activity was substrate specific for ATP and had a temperature optimum near 40 degrees C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K(+)-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K(+) > Rb(+) > Na(+) > Li(+) but when the concentration was raised to 50 millimolarity, the sequence changed to K(+) >/= Na(+) >/= Rb(+) > Li. The activity was not synergistically stimulated by combinations of Na(+) and K(+). The enzyme was insensitive to NaN(3), oligomycin, ouabain, and sodium molybdate but sensitive to N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.