Cytochromes of plant plasma membranes. Characterization by absorbance difference spectrophotometry and redox titration

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The cytochrome composition of plasma membranes (PM) obtained by phase partitioning of microsomal fractions from spinach leaves (Spinacea oleracea L. cv. Medania), cauliflower inflorescences (Brassica oleracea L.), sugar beer leaves (Beta vulgaris L.) and barley (Hordeum vulgare L. cv. Kristina) roots and leaves was characterized by absorbance difference spectrophotometry at different reducing conditions at 20 and – 196°C, by redox titration, and by heme staining of polypeptide bands after lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS‐PAGE). The location of the α‐bands in the difference spectra and the loss of heme after treatment with LDS indicated that predominantly cytochromes of the b‐type were present in all species tested. The total concentration of cytochrome was ca 0.35 nmol (mg protein)−1. The main component (ca 70% of total) was completely reduced by ascorbate and partly by NADH and had a midpoint potential of ca 150 mV. At – 196°C, ascorbate reduction revealed a symmetrical α‐band at ca 557 nm with PM from spinach leaves, cauliflower and sugar beet leaves, but with barley root and leaf PM ascorbate reduction resulted in an asymmetrical α‐band (shoulder at 552, maximum at 559 nm). In the dithionite‐reduced minus ascorbate‐reduced spectrum at –196°C a split α‐band (552 + 558 nm) was seen with PM from all species. This minor component had a midpoint potential of ca – 50 mV and is probably identical to cytochrome b5, the presence of which would explain the relatively high NADH‐cytochrome c reductase activities observed with plant PM. With PM from cauliflower, CO‐difference spectra indicated that cytochromes P‐420 and P‐450 were present at concentrations up to 0.06 and 0.03 nmol (mg protein)−1, respectively. Visualization of cytochromes by heme staining after LDS‐PAGE was complicated by endogenous peroxidase activity and by loss of heme during solubilisation. A presumptive b‐cytochrome (heme‐stained band at 94 kDa) was only detected with barley leaf PM.