CYTOCHROME F FROM PLANTS AND CYANOBACTERIA
Abstract
Existing methods of isolation and purification of cytochrome f were modified to give extraction yields of close to 100 percent and purification yields of 80 to 90 percent. This procedure was successfully applied to cyanobacteria, a eukaryotic alga, and higher plants. In addition schemes were developed to obtain both monomeric and aggregated cytochrome f from the same organism. The first 81 residues of Spirulina maxima cytochrome f and the first 78 residues of spinach cytochrome f were determined by automated sequencing of fragments produced by chemical and proteolytic digestion. Fifty-nine of the eighty-one residues of the cyanobacterial cytochrome f are conserved in the eukaryotic protein. The heme binding site is located in this region. A low potential cytochrome, cytochrome c(,550), occasionally found in various species of cyanobacteria and eukaryotic algae and considered to be a proteolytic product of cytochrome f was isolated and purified by conventional and high performance liquid chromatography. N-Terminal amino acid sequence analysis proves the distinctness of this protein from cytochrome f. Indeed, cytochrome c(,550), the high potential cytochrome c(,553), and cyanobacterial cytochrome f are not antigenically related as determined by immunodiffusion experiments using antibodies raised against each cytochrome. Modification of intact and disrupted spinach thylakoid membranes with diazonium benzene sulfonic acid (DABS) was employed to investigate the location of plastocyanin and cytochrome f relative to the bilayer. The labeling patterns for plastocyanin and cytochrome f were dependent on the state of the membranes. Similar patterns were observed with thylakoids either incubated in the dark or exposed to light. Comparison with data obtained earlier for the labelling of cytochrome oxidase under similar conditions suggests that parts of the cytochrome f polypeptide chain are exposed at both sides of the photosynthetic membrane.
Degree
Ph.D.
Subject Area
Biology
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