Plasma Membrane-associated Adenosine Triphosphatase Activity of Isolated Cortex and Stele from Corn Roots

An ATPase preparation, presumed to be associated with plasma membrane due to the coincidence on isopycnic gradients of ceUulase and ,8-glucan synthetase at high substrate, has been isolated from the epidermal and mesophyil of tobacco leaf. The ATPase from both tissues was found to prefer ATP over other nucleotides. The pH optimum was 7.0 in the presence of 3 millimolar MgCI2 and pH 6.5 in the presence of 3 millimolar MgCI2 and 100 milHimolar KCI. Monovalent ion stimulation patterns of the ATPases from these tissues were found to differ and ion accumulation patterns in these tissues reflect this difference: mesophyU accumulated roughly equal amounts of Na+ and Rb and its plasma membrane ATPase is also equaly stimulated by these ions; on the other hand, epidermal ATPase preparations showed a stronger stimulation by Rb+ than Na+ and this tissue was found to accumulate Rb' in preference to Na+. Abscisic acid and fusicoccin affected both ATPase activity and ion uptake, the former inhibiting and the latter stimulating these parameters. These data support the hypothesis that the epidermal plasmalemma ATPase is involved in stomatal opening.

supporting

confidence: 66%

mentioning

confidence: 99%

Differential Ion Stimulation of Plasmalemma Adenosine Triphosphatase from Leaf Epidermis and Mesophyll of Nicotiana rustica L

Lurie¹,

Hendrix²

1979

“…Kinetic data (29) for K+ stimulation of ATPase activity from the cortex of corn roots were similar to those for primary roots, i.e. consistent with the multiphasic concept.…”

Section: Resultssupporting

confidence: 64%

“…Similar data from the stele approximated the Michaelis-Menten equation, however. This indicates either an inherent difference between the enzyme in the two tissues or, more simply, a contamination of the stelar preparation by mitochondrial ATPase (29). A K+-stimulated ATPase of low molecular weight has recently been solubilized and purified from corn roots (5).…”

Section: Resultsmentioning

confidence: 99%

Stimulation of Plasmalemma Adenosine Triphosphatase from Oat Roots by Inorganic and Organic Cations: Concentration-Dependence, Selectivity, and Sites

Håvarstein¹,

Nissen²

1981

K-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 m;limolar KCI. The phases obeyed MlchaeLs-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 nillilar. Stimulation by alkali cations was in the order K+ > Rb+ > Na+ > Cs' > Li' or in a closely related sequence. The specificity reflected differences in ,,.,, not in affinity (K.'). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 mllmolar appeared monophasic rather than biphasic. Substitution on the quatemary nitrogen of the amino alcohols decreased their effectiveness, as did extension and brhing of the chain. Stimulation was maximal at about pH 7 both for K' and choline.The kinetics of K' stimulation are multiphasic, not cooperative, as was also found for uptake. The ATPase is also stimulated by organic cations, but the difference in kinetics indicates the existence of separate sites for stimulation and transition.Membrane-bound ATPases have been implicated in the transport of ions in plants (17,18,30). Particularly close correlations exist between a K+-stimulated ATPase and uptake of K+ across the plasmalemma (13,27,28). The role of this enzyme in ion transport remains unclear, however (25, 43).The present paper examines some unresolved questions concerning this relationship. The complex kinetics of ion uptake and ATPase stimulation have been described variously as cooperative (17,27,28) MATERIALS AND METHODS Seedlings of oat (Avena sativa L. cv. Titus) were grown in 0.5 mM CaC12 for 6 to 8 days at 16 to 22 C in the dark (40).A plasmalemma-enriched membrane fraction was isolated as described by Hodges and Leonard (19) with some minor modifications (32). The excised roots were ground with a mortar and pestle in a solution of 250 mm sucrose, 3 mm EDTA, 2.5 mm DTT, and 25 mm Tris-Mes buffer (pH 7.2). The filtered homogenate was centrifuged successively at 13,000g for 15 min and 40,000g for 30 min. The 40,000g pellet was suspended in 34% sucrose (w/w) and layered onto a discontinuous gradient consisting of 20 ml of 45% sucrose (w/w), 8 ml of 40%o sucrose, and 8 ml of 34% sucrose. All sucrose solutions contained 1 mm DTT and 1 mM Tris-Mes (pH 7.2). The gradient was centrifuged for 2 h at 80,000g in a Spinco SW 27 rotor, and the membrane fraction was collected at the 34/ 40% sucrose interface. For most experiments the membrane fraction was frozen at -18 C and used the following day (cf. Figs. 1 and 2).ATPase activity was measured in a l-ml volume containing 3 mM ATP (Tris salt, pH 6.5), 30 mm Tris-Mes (pH 6.5), 3 mM MgSO4, varying concentrations of monovalent inorganic or organic cations (as chlorides), and 10 to 25 ,tg of membrane protein.Reactions, initiated by the addition of ATP, were carried out at 38 C in a shaking water bath for 30 to 45 min, depending on the amount of protein. (ATPase activity was linear for at least 1 h, and the ATP concentrations remained essentially constant.) Pi released was de...

“…Vol. 69,1982 drial-associated activity (8). Alternatively, the PM-associated ATPase may be inhibited by vanadate (16), and the vanadatesensitive activity could offer a better way to mark PM in plant cell homogenates.…”

Section: Resultsmentioning

confidence: 99%

Effects of Centrifugal Force and Centrifugation Time on the Sedimentation of Plant Organelles

Nagahashi

Hiraike²

1982

The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 80OOg or 10,OOOg for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker, however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.Inasmuch as mitochondria, RER, and pM2 have similar densities in sucrose gradients. it is necessary initially to separate the mitochondria from microsomes by differential centrifugation. A range of centrifugal forces, from 4,600g to 20,000g (1)(2)(3)(4)(5)14), has been used to achieve this separation; however, these crude mitochondrial fractions also contain substantial amounts of Golgi membrane, ER, and PM (6,9,12,15). These results can be viewed either as a loss of microsomal vesicles, if one is attempting to purify microsomes, or as a contaminated mitochondrial fraction.A recent investigator (3) has used a crude mitochondrial fraction (l0,OOOg pellet) as a direct source of plasma membranes; however, further separation of these two organelles proved to be difficult. A Ficoll density gradient was used, and it provided limited success, inasmuch as the plasma membrane markers were only slightly enriched after centrifugation and there was still considerable overlap between mitochondria and PM markers (3). These results exemplify the need for initial separation of the mitochondria from microsomes prior to density-gradient separation.This report is concerned with the determination of the optimal differential centrifugation conditions (centrifugal force and length of time) that produce the least amount of cross-contamination between mitochondria and microsomes. Only a few plant investigators (2,5,9,12) Enzyme Assays. The K+-stimulated ATPase activity was determined as described previously (9). To retain the K+-stimulated ATPase activity, corn roots were homogenized in DTT or dithioerythritol. When ,B-mercaptoethanol was used in the grinding medium, Cyt c oxidoreductase activities were maintained, but the K+-stimulated ATPase activity was very poor. Cyt c oxidase activity and NADH Cyt c reductase activity were determined as described by Hodges and Leonard (4). NADH Cyt c reductase activity was assayed in the presence and absence of antimycin A (10). Proteins were estimated according to Lowry et al. (I 1), and all enzyme assays were performed with 20 to 40 ,ug of membrane protein. RESULTSDifferential Centrifugation between 1,000g and 80,000g. Centrifugation at 3,000g for 45 min only sedimented 75% of the Cyt c oxidase activity (Fig. 2). Although we did not centrifuge for longer periods of time at 3,000g,...