Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus

Abstract

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of C. aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a NaI gradient centrifugation were used in isolating crytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high (3000 W m-2), medium (200 W m-2) or low (7 W m-2) light-grown cells had near IR absorption bands at 866, 808 and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center and reaction center per mg of cytoplasmic membrane protein was constant. No Bchl c was present. Five respiratory enzyme activities were measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent MW of 8000. SDS-PAGE of purified chlorosomes did not contain the 8000 MW band but revealed only 3 distinct protein bands with MW of 15,000, 12,000 and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. Thus, the components of the photosynthetic apparatus in the cytoplasmic membrane of C. aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.