Comparison of two methods for the preparation of a membrane fraction of cauliflower inflorescences containing a blue light reducible b ‐type cytochrome

Presumptive plasma membrane fractions containing a light‐sensitive flavocyto–chrome b ‐protein complex from cauliflower inflorescences were isolated by two different procedures. In the first procedure a density gradient centrifugation was used, while in the second the separation was carried out using an aqueous polymer two phase system based on the specific surface properties of the membranes. This latter method is much faster and its yield is as good as a purification on a linear sucrose density gradient in terms of light inducible cyt b reduction. When the LIAC‐containing membranes obtained through a sucrose gradient are further purified on an Urografin gradient, the presence of contaminating cyt b is shown. The distribution of this b type cytochrome, showing no redox change upon illumination, is similar to the distribution as a NADH dependent and antimycin A resistant cytochrome c reduc–tase, a marker enzyme for endoplasmic reticulum. Measurable mitochondrial contamination is indicated by cytochrome oxidase activity. Both contaminants were less pronounced in the fractions obtained after Urografin gradient centrifugation of the membranes prepared by the two phase system method. The isolation procedure based upon the use of the two phase aqueous polymer system allows more rapid preparation of LIAC‐containing presumptive plasma membranes than that obtained with the sucrose density gradient centrifugation. It also yields a purer preparation.