Membrane-bound Adenosine Triphosphatase Activities of Oat Roots

Abstract

Homogenates of oat (Arena satira cv. Goodfield) roots contained at least five membrane-associated adenosine triphosphatase (ATPase) activities. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density, pH optima, sensitivity to monovalent salts, and substrate specificity.A membrane fraction sedimenting at low centrifugal force (13,000g) contained two ATPase activities at pH 9.0. One membrane ATPase was coincident with cytochrome c oxidase activity and had a density of 1.18 grams per cubic centimeter. This membrane system was identified as mitochondria. The other pH 9.0 ATPase in this fraction occurred at a density of 1.16 grams per cubic centimeter. The identity of this membrane is unknown.Three additional ATPases were in a membrane fraction sedimenting at high centrifugal forces (13,000-80,000g). One membrane ATPase coincided with NADH-cytochrome c reductase activity, had a density of about 1.09 grams per cubic centimeter, and was equally active at pH 6.0 and 9.0. A second membrane ATPase of the 13,000 to 80,000g fraction had a density of 1.13 grams per cubic centimeter and was more active at pH 9.0 than at pH 6.0. A third membrane ATPase had greater activity at pH 6.0 than at pH 9.0, and the membrane had an apparent density of 1.17 grams per cubic centimeter on the sucrose gradient. This ATPase was especially sensitive to KCI. The identity of the membranes which contain ATPases is discussed in relation to the distribution of other enzymes on the gradient.We have previously described an ATPase from roots that was associated with membranes, required Mg2+, and was stimulated by monovalent salts in general but with KCI and RbCl giving the greatest activation (8). A For the above concept to be tenable, the ATPase must be associated with either the plasma membrane or tonoplast since these membranes are the major sites of active ion transport in higher plants (20,23). Indeed, histochemical studies have shown that both the plasma membrane (11,24) and tonoplast (12,24) have ATPase activities. However, membrane systems of such structures as mitochondria, nuclei, endoplasmic reticulum, and Golgi also possess ATPase activity (12,24). Hence, in vitro assays of ATPase activity employing a crude preparation of membranes (i.e., a differentially centrifuged fraction) may measure several different ATPases. It would be desirable to know how many membrane-associated ATPases can be distinguished in cell extracts and to identify the various membrane systems containing these enzymes. This was the purpose of the investigations reported here. We show that at least five ATPase activities can be distinguished by density gradient centrifugation of membrane fractions from oat roots. One of these ATPases is associated with mitochondria, and another is associated with plasma membranes (16). The latter enzyme is especially sensitive to KCl. The membranes containing the other ATPases are unidentified. MATERIALS AND METHODSPlant Material. Oat seeds (A vena sativa cv. Goodfield) were suspend...

Presence of Two Different Membrane-bound, KCl-stimulated Adenosine Triphosphatase Activities in Maize Roots

“…The distribution of enzyme activities in fractions prepared according to Scheme A are given in Table I. The highest specific activity of Mg2+-ATPase at pH 5.5 ATPases FROM I KCl-stimulated ATPase are lower than those found in oat root membrane fractions (6,10,12), they are comparable to previously reported values for the activity of this enzyme in maize root membrane fractions (4,11,17,24).…”

“…The properties of the ATPase found in the light Ficoll fraction implicated it in ion transport (24). Leonard et al (10) also found a small peak of KCIstimulated ATPase activity from oat roots at low buoyant densities but regarded it as less significant than the activity associated with the plasmalemma.…”

Evidence for a Cl−-Stimulated MgATPase Proton Pump in Oat Root Membranes

“…The rate of ATP-hydrolysis by plant MgATPases is dependent upon the presence of Mg ions and is stimulated by the presence of KCI (10,11). Figure 3 (10,18).…”

Cadmium Alteration of Root Physiology and Potassium Ion Fluxes

“…The surprising finding is that respiration occurs at 50%o of the control rates after a 24-hr (Table IV) which has previously been correlated with K+ uptake (6). This ATPase is bound to a membrane fraction; one component of this fraction is the plasmalemma (15). In animal cells I mM Cd2+ inhibited mitochondrial ATPase and plasma membrane ATPase to 70% of control rates and after a 2 mm Cd2" treatment, 50% of control rates remained (17).…”

“…The (15). The membrane pellet (13,000-80,000g) was suspended in 0.25 M sucrose and assayed according to the procedure of Leonard et al (15) at 30 C (pH 6) with Tris-ATP, in the presence or absence of I mm CdSO4. Potassium Uptake.…”