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Isolation of plasma membrane and binding of the Ca 2+ antagonist nimodipine in Chlamydomonas reinhardtii
Author(s) -
Dolle Reiner
Publication year - 1988
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1988.tb09185.x
Subject(s) - membrane , chemistry , dissociation constant , chromatography , microsome , chlamydomonas reinhardtii , biochemistry , enzyme , receptor , mutant , gene
Chlorophyll‐free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two‐phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 m M NaCI, 0 33 M sucrose and 5 m M potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg 2+ ‐ATPase (EC 3.6.1.3). was enriched 9‐fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol‐rich phase. Contamination from intracellular membranes was minimal. K*, Mg 2+ ‐ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X‐100 stimulated the activity 3‐fold. [ 3 H]‐Nimodipinc was employed to characterize 1,4‐dihydropyridine‐specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (K D = 9 n M ) was located on the plasma membrane and a second binding site with lower affinity (K D = 36 n M ) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was B max = 440 and 470 fmol (mg protein) ‐1 , respectively. [ 3 H]‐Nimodipinc binding was inhibited by (±) verapamil and stimulated by D‐ cis ‐diltiazem in both fractions. Moreover, ethyle‐neglycol‐bis(2‐aminoethylcther)‐N, N'‐tetraacctic acid (EGTA) inhibited [ 3 H]‐nimo‐dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl 2 .