Distribution of ATPases in wheat root membranes separated by phase partition

Abstract

The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots (Tritium aestivum L. cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g, 10,000 g, 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two‐phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter‐current distribution id a 6.3/6.3% two‐phase system. Protein and activities of Ca2+, Mg2+, and Mn2+ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured. The partition of ATPase activities stimulated by Ca2+, Mg2+ or Mn2+ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3‐system was selected for further separation of the membranes in the 30 KP fraction by counter‐current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.

Plasma Membranes from Oats Prepared by Partition in an Aqueous Polymer Two-Phase System

“…This small peak was presumed to be mainly plasma membranes. Similar results have been obtained with corn coleoptiles (30) and wheat roots (20). In these cases, LIAC was used as marker for the plasma membrane.…”

“…Thus, intact protoplasts from Digitaria sanguinalis partition in the upper phase, while chloroplasts and cell debris collect at the interface (12); and plasma membranes have been prepared from several species and tissues utilizing this high partition ratio for the plasma membrane (20,30). Similarly, in the present study on oats, plasma membrane fractions with high partition ratios could be isolated from both roots and shoots.…”

“…Oats (Avena sativa L. cv Brighton) were grown in darkness as described elsewhere for wheat (20). After a week, the seedlings were harvested and divided into shoots and roots.…”

“…The phase composition earlier found to be optimum for purification of wheat root plasma membranes (20) was used: 6.3% (w/w) dextran T 500, 6.3% (w/w) PEG 4000, 0.25 M sucrose, 5 mm K-phosphate, pH 7.8 (5°C). A phase system of 200 g was prepared in a separatory funnel, by weighing from stock solutions.…”

“…fraction with very low contamination of mitochondria and ER (20,30). These plasma membranes were purified from microsomal fractions by partition in a dextran-PEG two-phase system.…”