Chloroplast Membrane Proteins: II. SOLUBILIZATION OF THE LIPOPHILIC COMPONENTS

Abstract

Abstract The membrane of spinach chloroplast grana has been solubilized by a method which does not involve use of detergents. This method is based on the fact that most of the membrane components are soluble in acidic ethanol. The alcoholic extract is chromatographed on a hydroxylapatite column and the pigments are removed quantitatively by washing with alcohol. The membrane proteins can then be eluted by successive washing with phosphate buffer and dilute ammonium hydroxide. Some protein remains on the column and requires sodium dodecyl sulfate for elution. The major component of the membrane is eluted in the ammonia fraction, and is freely soluble in aqueous buffers at pH 7.5. This protein was shown to bind β-carotene. None of the major chloroplast proteins are glycoproteins. The fraction which was insoluble in acidic alcohol had a bright pink color due to the presence of β-carotene. This complex was highly insoluble, even in high sodium dodecyl sulfate concentrations unless the β-carotene was first removed by extraction with an organic solvent. This complex was shown to be a precipitate of β-carotene and several peptides.