UTILIZATION OF ENZYME MARKERS TO DETERMINE THE LOCATION OF PLASMA-MEMBRANE FROM PISUM EPICOTYLS ON SUCROSE GRADIENTS

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. ''Alaska'') epicotyls were fractionated and examined for marker enzyme activity. The coincidence of 3 reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K+-stimulated Mg2+-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with EM evidence reported by G. Shore and G. Maclachlan, indicates that plasma membrane of pea epicotyl has a buoyant density of .apprx. 1.13 g/cm3. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10-1.11 g/cm3), Golgi (1.12 g/cm3) and mitochondria (1.18 g/cm3). Other recent literature indicates that the 1.13 g/cm3 buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. The data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g., glucan synthetase and K+-stimulated Mg2+-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm3 density, but with particles of higher densities as well). Particulate cellulase was associated only with particles with equilibrium densities of .apprx. 1.13 g/cm3. Cellulase proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm3 particulate components with minimal contamination by particles of higher densities.