Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue

Abstract

Washing of excised corn (Zea mays L., variety WF9xM14) root tissue is accompanied by an increase in (Mg2+ + K+)-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed.In the preceding paper (25) we describe the increase in absorption rates of phosphate, and of some other ions and solutes, which accompanies the washing of roots from seedlings grown under highly aerobic conditions. After an inductive lag period of about 30 min, there is a rapid development of phosphate absorption rate without significant change in respiration rate.Inhibitor studies indicate that RNA and protein synthesis are involved. However, no increase in membranes with washing could be found by electron microscopy or phospholipid analysis. Therefore, "transport" enzymes synthesized must be added to existing membranes. Since the apparent Km values for '2P and '6Rb absorption do not change, it would appear that existing carrier systems must be augmented or activated.One enzymatic system which is being explored for a possible role in ion transport is membrane-bound ATPase (1, 4, 5, 7-10, 12, 15, 17, 22, 29, 33 Incorporation and Distribution of "4C-Leucine. Root segments were incubated in 500-mg lots for 15 min in 10 ml of 0.2 mM CaCi2 containing 0.1 ,uc/ml of uniformly labeled 4C-leucine (316 mc/mmole). After being rinsed for 30 sec in ice-cold water and for 20 min in ice-cold unlabeled 1 mM leucine, the tissue was ground with a conical glass homogenizer in 0.2 M sucrose. The homogenate was successively centrifuged at 2,000g for 10 min (cell wall and nuclei), 12,000g for 20 min (mitochondria), and 80,000g for 60 min.The pellets were washed by resuspension and recentrifugation and were suspended in 0.2 M sucrose. Protein was precipitated from the supernatant, first in 50% saturated (NH4),-SO4 and finally in 8% trichloroacetic acid. Each fraction was washed by resuspension and centrifugation and finally was suspended in 0.2 M sucrose. Aliquots of the pellets, precipitates, and final supernatant were assayed for radioactivity.Protein content was determined colorimetrically (27

Isolation of Plasma Membranes from Corn Roots by Sucrose Density Gradient Centrifugation

“…At pH 9, ATPase activity was highest in the 1,000 to 8,000g fraction and in the mitochondrial fraction from the sucrose gradient. In general, ATPase activities were comparable to those previously reported for membrane fractions from corn roots (18), but were substantially higher than those reported by Wyn Jones and colleagues (15,38) for a plasma membrane-rich fraction from primary roots of this same variety of corn. One reason for this difference is that they assayed ATPase at 30 C, which is well below the optimum of 40 C for the plasma membrane ATPase of corn roots (unpublished results).…”

“…As expected (17,18), all membrane fractions contained K+-stimulated ATPase activity at both pH 6.5 and 9 (Tables I and II). At pH 6.5, the highest K+-stimulated activity was in the plasma membrane fraction (Table II).…”

Increase in Electrogenic Membrane Potential with Washing of Corn Root Tissue

“…Energy for the pump might arise from an ATPase, provided that the membrane-bound system has certain properties (7) and indeed, there is an increase with washing in ATPase of the microsome fraction (6). The problem here is that the increase in ATPase parallels that of phosphate absorption, not that of the electrogenic PD (6; cf.…”

Action of Protein Synthesis Inhibitors in Blocking Electrogenic H+ Efflux from Corn Roots

“…The It has been repeatedly demonstrated in a wide variety of plant tissues that inhibitors of protein synthesis, such as cycloheximide, will also inhibit energy-linked ion absorption, but the nature of this inhibition is not resolved. In tissues where ion-absorption rates are increasing due to washing or aging (13,17,25,26), the inhibition might be reasonably attributed to blocked synthesis of those enzymes required to augment transport rates. In other cases, however, there have been suggestions that protein synthesis inhibitors may have secondary or side effects, possibly interfering with metabolic energy supply (9,14,22 tions, also blocked IAA-induced growth and its associated increases in respiration, H+ efflux, and cell potential.…”