Isolation and Identification of Plasma Membrane from Light-Grown Winter Rye Seedlings (Secale cereale L. cv Puma)

Abstract

An effective method for the isolation of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma) was established using a liquid two-polymer phase separation. The conditions for the specific partition of plasma membrane into the polyethylene glycol-enriched upper phase were examined, including variations in the polymer concentration, buffer system, pH, and NaCl addition in the phase partition system. The most effective phase partition system for the isolation of plasma membrane from winter rye consisted of 5.6/5.6% (w/w) polyethylene glycol 4000/dextran T500 in 0.25 molar sucrose-10 millimolar potassium phosphate-30 millimolar NaCl (pH 7.8), repeated once. When the isolated plasma membrane was centrifuged on a linear sucrose density gradient, a single band was found at the 34% (w/w) sucrose layer (1.141 grams per cubic centimeter) which co-fractionated with the pH 6.5-ATPase. Identification of plasma membrane was performed by the combination of phosphotungstic acid-chromic acid stain and specific binding of N-1-naphthylphthalamic acid. Based on morphometrical observations after phosphotungstic acid-chromic acid stain, the isolated plasma membrane consisted mostly of vesicles of high purity. The isolated plasma membrane also showed extremely high specificity for N-1-naphthylphthalamic acidbinding, 10-fold higher than other membranes. It was also confirmed that there is a distinct difference in properties between plasma membrane and other membranes. The endomembranes such as from chloroplasts, mitochondria, and endoplasmic reticulum were observed to be highly sensitive to Zn²⁺ ion and lower pH, which resulted in an abrupt aggregation of membranes. On the contrary, plasma membrane was very stable to these treatments and no aggregation was observed. These unique properties of isolated plasma membrane are generally observed in a wide variety of plant species and can be utilized for the assessment of the purity of preparations of isolated plasma membranes and for their identification.