Cytosolic factors in bovine neutrophil oxidase activation. Partial purification and demonstration of translocation to a membrane fraction

Journal Article · Tue Mar 06 04:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00461a004· OSTI ID:6940740

Doussiere, J; Pilloud, M C; Vignais, P V ^[1]

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1. Centre d'Etudes Nucleaires, Grenoble (France)

The O{sub 2}{sup {center dot}{minus}}-generating oxidase of bovine neutrophils is activated in a cell-free system consisting of a particulate fraction enriched in plasma membrane and containing the dormant oxidase, a high-speed supernatant from neutrophil homogenate (cytosol), Mg ions, GTP{gamma}S, and arachidonic acid. The cytosolic components participating in the activation of the membrane-bound oxidase have been investigated. These components were resolved into several active peaks by Q Sepharose chromatography. Partial purification of two active fractions containing a limited number of proteins of 65, 56, 53, and 45 kDa was achieved by gel filtration of cytosol on Ultrogel AcA44, followed by chromatography on hydroxylapatite and Mono Q. The specific oxidase-activating potency of these partially purified fractions was 6-8-fold higher than that of crude cytosol. These data indicate that oxidase activation requires more than one cytosolic component to be activated. To check whether translocation of cytosolic proteins to the membrane occurred concomitantly with oxidase activation, use was made of radiolabeled cytosolic proteins. Cytosol was treated with phenyl({sup 14}C)isothiocyanate (({sup 14}C)PITC). Translocation was studied under conditions in which production of O{sub 2}{sup {center dot}{minus}} was largely modulated by varying the amount of arachidonic acid added to the cell-free system. Maximal oxidase activation with optimal concentration of arachidonic acid resulted in the selective translocation of labeled cytosolic proteins of 65, 53, 45, and 17 kDa to the membrane.

• OSTI ID: 6940740
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:9; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOLOGICAL EFFECTS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBON 14 COMPOUNDS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
DOMESTIC ANIMALS
ENZYME INDUCTION
ENZYMES
GENE REGULATION
ISOTHIOCYANATES
LABELLED COMPOUNDS
LEUKOCYTES
MAMMALS
MATERIALS
MEMBRANES
NEUTROPHILS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
PHAGOCYTOSIS
PURIFICATION
RADICALS
RUMINANTS
SUPEROXIDE RADICALS
TRANSLOCATION
VERTEBRATES