Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.
The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.
Hancock
J T
JT
Department of Biochemistry, University of Bristol Medical School, U.K.
Maly
F E
FE
Jones
O T
OT
D001402:B-Lymphocytes / Q000201:enzymology*; D002051:Burkitt Lymphoma / Q000201:enzymology; D002460:Cell Line; D003573:Cytochrome b Group / Q000378:metabolism; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; C035130:cytochrome b245; D013481:Superoxides; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme