Characterization of a K⁺-Stimulated Adenosine Triphosphatase Associated with the Plasma Membrane of Red Beet

Abstract

A membrane fraction enriced with a mapesium-dependent, monovalent cation-stimulated ATPase was Isolated from red beet (Beta vlgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fractio was distinct from eadpamcreticuum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with tis fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mge and K+. The acthiity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with MglATP followed a simple MichaelisMenten relationship. However the kinetics of K+-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 milhmolarity, ATPase was stimulated in the sequence K+ > Rb+ > Na+ > Li+ but when the concentration was raised to 50 nflhlmolarity, the sequence changed to K+ a Na+ a Rb+ > Li. The activity was not synergisticaly stimulated by combinations of Na+ and K+. The enzyme was iosensitive to NaN3, oligomycin, ouabain, and sodium molybdate but sensitive to N,f-icyclobexylcarbodllmlde, diethyistilbestrol, and sodim vanadate. Based on the shiilarity 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.Plant storage tissue has provided a convenient system for the study of cellular ion transport. The use of storage tissue in ion flux studies has the advantage that transport can be considered for individual cells unaffected by long distance transport, growth, and photosynthesis (19). Ion transport in storage tissue has been well characterized (19 and references therein) and for red beet, ATP has been shown to be the energy source (18 membrane ATPase of red beet. First, beet storage tissue is available in bulk and thus potentially suitable for enzyme purification and for phosphorylation studies. Secondly, we were interested to know to what extent the unusual transport features of beet (19)-its halophytic nature, propensity for accumulation of sodium, and the large effect of pH on cation accumulation-might be related to the properties of the plasma membrane ATPase. While the ATPase activity in crude membrane fractions from this species (sugar beet) has been analyzed (6), the properties of this activity in purified plasma membrane fractions have not been characterized.In this paper, cell fractionation studies with red beet storage root are presented. A membrane fraction enriched with Mg-dependent, monovalent cation-stimulated ATPase activity distinct from ER, Golgi, and mitochondrial enzyme markers was prepared. The properties of the ATPase activity were similar to those reported for corn (12) and oat (8) root plasma membrane fractions. MATERIALS AND METHODSPlant Material. Red beet (Beta vulgaris L.) storage roots were purchased commercially...

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot

“…Since our goal was to use the sealed vesicle system for the study of sucrose transport, it was desirable to isolate the vesicles in the absence of sucrose. In preliminary experiments, the sugarbeet root homogenate was centrifuged at 13,000g prior to collection of microsomal membranes by centrifugation at 80,000g and following KI extraction of the microsomes (to diminish nonspecific phosphatase, see Briskin and Poole [5]), the sealed vesicles were recovered at a 10% (w/w) dextran gradient interface (25,26). It was found, however, that maximal enrichment ofH+-transporting activity on a protein basis relative to a reduction in contaminant marker enzyme activities (see below) occurred when the low speed centrifugation was reduced to 7,700g and the density of the dextran at the gradient interface was reduced to 6% (w/w).…”

“…This paper describes the isolation and characterization of the sugarbeet vesicles while in a subsequent paper (6), the sugarbeet vesicle system is used to examine the mechanism of sucrose transport. (5). Following incubation for 20 min on ice, the KI-treated membranes were centrifuged at 80,000g (32,500 rpm) for 30 min in an IEC A-237 rotor.…”

Analysis of factors controlling dinitrogen fixation and nitrogen release in soybean using pod removal, stem girdling, and defoliation

“…However the abruptness in the interruption of the flow of sugars to the root system could result in the rapid senescence or sharp reduction in root and nodule ATP concentrations. Enough evidence supports the fact that H + pumps which are integral parts of the plasma membrane, are driven by ATP (ATPases) (Briskin and Poole 1983) and are essential for its functional integrity (Poole 1978). Thus an abrupt interruption in the photosynthate supply to the roots could reduce the ATP levels which play an important role in the maintenance of the root membrane integrity (Elzbieta et al 1985).…”

1-Naphthyl Acetate-Dependent Medium Acidification by Zea mays L. Coleoptile Segments

“…This decrease in pH activates proton pumping at the plasmalemma, possibly shifting the cytoplasmic pH closer to the pH optimum of the pump. Plasmalemma ATPase activity showed a pH optimum close to 6.5 (1)(2)(3)8). Since cytoplasmic pH of plant cells tends to be in the 7.0 to 7.6 range (16,20 and references therein), this means that a decrease of cytoplasm pH may be expected to result in a significant increase in proton pump activity.…”