PURIFICATION AND CHARACTERIZATION OF TWO FORMS OF APOCYTOCHROME B FROM YEAST MITOCHONDRIA.
Item
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Title
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PURIFICATION AND CHARACTERIZATION OF TWO FORMS OF APOCYTOCHROME B FROM YEAST MITOCHONDRIA.
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Identifier
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AAI8112739
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identifier
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8112739
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Creator
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CHEN, YU-SHIAW.
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Contributor
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Diana S. Beattie
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Date
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1981
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Cytochrome b in a partially purified preparation from yeast mitochondria showed normal migration behavior during dodecyl sulfate electrophoresis in different concentrations of acrylamide. This protein migrated at a molecular weight of 31,000 when the preparation was heated in dissociation medium at 20(DEGREES), 37(DEGREES), 70(DEGREES) or 100(DEGREES)C when phenylmethanesulfonyl fluoride, the protease inhibitor, was present or absent and in dodecyl sulfate urea gels. By contract, cytochrome b in the intact mitochondrial membrane displayed anomalous migration behavior in gels of different acrylamide concentrations.;Two forms of apocytochrome b were purified by a procedure involving ammonium sulfate fractionation, Ultrogel filtration, minicon concentration, polyacrylamide gel separation and acetone extraction. Both proteins showed one single band with distinct molecular weight, protein I of 31,000 dalton and protein II of 29,00 dalton, upon polyacrylamide gel electrophoresis. Purified protein I and protein II showed similar antigenical response and identical fingerprints after limited proteolytic digestion. So far, this is a report about two species of cytochrome b, for the first time, purified to homogeneity.;Antisera against apocytochrome b-I and b-II were raised individually. The specificity of these antisera was established by (1) ouchterlony double diffusion test, (2) counter immunoelectrophoresis, (3) immunotitration of labeled mitochondrial extract with antiserum, (4) electrophoretic analysis of immunoprecipitates. Both apo-cytochrome b-I and b-II were demonstrated to be translated on mitochondrial ribosomes by labeling yeast cells in the presence of cycloheximide and immunoprecipitating with antiserum.;Both IgG fractions purified from antisera against apocytochrome b-I and b-II inhibited cytochrome c reductase activity of a mitoplast preparation, an inner membrane-matrix fraction and a reverse-sided submitochondrial particles preparation suggesting the transmembraneous occurence of both apocytochromes b in the mitochondrial inner membrane. Radioautographical analysis of immunoprecipitates from diazotized (('125)I) iodosulfonate, the membrane non-penetrating reagent, labeled mitochondria and submitochondrial particles also revealed that both apocytochromes b span the mitochondrial inner membrane.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
