Abstract
Chloroplastic envelope membranes isolated from pea (Pisum sativum) leaves are rich in carotenoids, containing approximately 2 micrograms of carotenoid mg-1 protein. We report here that envelopes can be surfactant-solubilized while maintaining association of carotenoids with protein components of the membrane. Treatment of isolated chloroplastic envelope membranes with 0.5% Deriphat 160 (N-lauryl-beta-imminodipropionate) causes general solubilization but preserves an envelope sub-membrane fragment which is fractionated by centrifugation in a sucrose gradient and by chromatography on a column of DEAE-Sephacel. The isolated submembrane complex contained five major proteins with M(r) values equivalent to 75,000, 36,000, 34,000 17,500, and 14,500. Spectroscopic and chromatographic analyses revealed that the complex contains violaxanthin and at least one other carotenoid. Carotenoid content of the fractionated complex was estimated as 4.8 micrograms mg-1 protein. Immunoblot analysis reveals that the constituent proteins of this complex are derived from the chloroplastic outer envelope membrane. These data suggest that at least some of the carotenoids of the chloroplastic envelope may be organized by apoproteins.
Highlights
Fractions of roplasts or from purified envelope membranes and fractionaapproximately 1 ml were collected as the column was successively washed with 4-ml aliquots of25 mM MOPS-NaOH (pH 7.0), 0.1% Deriphat 160 containing 0, 0.1,0.2,0.3, 0.4, or 0.5 M NaCl. This preparation protocol has been scaled up 3-fold with identical results by using six sucrose gradients, but employing the same size DEAESephacel column
Chloroplasticenvelope membranes isolated from pea thylakoid membranes. Because of their solubility in solvents (Pisum sativum) leaves are rich in carotenoids, con- such as methanol and acetone, carotenoids parreesumably in taining approximately 2 pg of carotenoid mg” protein. noncovalent association with any apoproteins, and previous
Immunoblot analysis tionation of a surfactant-stable particle containing a subset reveals that the constituent proteins of this complex of the chloroplastenvelope carotenoids. This carotenoid-conarederived from the chloroplastic outer envelope tainingsub-membraneparticleis derivedfrom the chloromembrane
Summary
Fractions of roplasts or from purified envelope membranes and fractionaapproximately 1 ml were collected as the column was successively washed with 4-ml aliquots of25 mM MOPS-NaOH (pH 7.0), 0.1% Deriphat 160 containing 0, 0.1,0.2,0.3, 0.4, or 0.5 M NaCl. This preparation protocol has been scaled up 3-fold with identical results by using six sucrose gradients, but employing the same size DEAESephacel column. Thedry pigment was dissolved in ethanol or methanol and quantitated using an extinction coefficient of 142 mM"cm" a t 440 rated by spectralcharacterization as published previously [25].Fractionation of pigments extracted from purified chloroplastic envelopes revealed two major peaks of similar area eluting with the retention times of violaxanthin and lutein/
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
