Characterization of membrane-bound electron transport enzymes from castor bean glyoxysomes and endoplasmic reticulum.
Membranes purified from castor bean endosperm glyoxysomes by washing with sodium carbonate exhibited integral NADH:ferricyanide and NADH:cytochrome c reductase activities. The enzyme activities could not be attributed to contamination by other endomembranes. Purified endoplasmic reticulum membranes also contained the redox activities; and marker enzyme analysis indicated minimum cross contamination between glyoxysomal and endoplasmic reticulum fractions. The glyoxysomal redox activities were optimally solubilized at detergent to protein ratios (weight to weight) of 10 (Triton X-100), 50 (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 100 (octylglucoside). Detergent in excess of the solubilization optimum was stimulatory to NADH:ferricyanide reductase and inhibitory to NADH:cytochrome c reductase. Endoplasmic reticulum redox activity solubilization profiles were similar to those obtained for glyoxysomal enzymes using Triton X-100. Purification of the glyoxysomal and endoplasmic reticulum NADH:ferricyanide reductases was accomplished using dye-ligand affinity chromatography on Cibacron blue 3GA agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of NADH:ferricyanide reductase preparations purified by rate-zonal density gradient centrifugation, affinity chromatography, and nondenaturing electrophoresis of detergent-solubilized glyoxysomal and endoplasmic reticulum membranes consistently displayed 32- and 33-kDa silver-stained polypeptide bands, respectively.
Department of Biological Sciences, George Washington University, D.C. 20052.
D G
DG
Luster
M I
MI
Bowditch
K M
KM
Eldridge
R P
RP
Donaldson
D002367:Ricinus communis / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000302:isolation & purification; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010947:Plants, Toxic; D012278:Ricinus / Q000201:enzymology*; D012995:Solubility
D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D009245:NADH Dehydrogenase