ABSTRACT. In intact neutrophils, phorbol ester treatment activates the respiratory burst oxidase, the enzyme responsible for O2-production by phagocytes. This effect is thought to be dependent on protein kinase C and on the phosphorylation of p47phox. In this paper, we report that protein kinase C activates the respiratory burst oxidase in a cell-free system consisting of isolated neutrophil cytosol and membrane. Oxidase activation required a highly active protein kinase C, recombinant p47phox and ATP, and was inhibited by the protein kinase C inhibitors H-7 and GF-109203X. Partial depletion of cytosolic ATP by dialysis reduced oxidase activation by over 50%. In contrast, neither protein kinase C inhibitors nor ATP depletion affected oxidase activation by SDS. These findings strongly suggest that in the cell-free system, the oxidase can be activated by the phosphorylation of p47phox.

Keywords: neutrophils, respiratory burst oxidase, protein phosphorylation, protein kinase C.

Reprint requests to: Bernard M. Babior, M.D., Ph.D., Department of Biochemistry, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037 USA, phone (619) 554-8258, fax (619) 554-6223, e-mail: babior@scripps.edu.

ABSTRACT. Selective hybridization of small intestine and liver cDNA libraries was carried out using yeast artificial chromosomes (YACs) surrounding D6S105, the microsatellite that appears to be close to the gene for hereditary hemochromatosis (HFE). Of 14 candidate probes hybridizing with these YACs, only one, designated LD5-1, detected abnormalities in Southern blots of patients with hemochromatosis. Two different abnormalities were detected in 3 of 55 patients with hemochromatosis with the LD5-1 probe, and one of these was detected in one of 44 normal subjects. The gene that hybridizes with this probe is located about 300-400 kb centromeric of D6S105. It is transcribed into mRNA that is about 8.5 kb in length in many tissues, including peripheral blood leukocytes. The available sequence indicates that it codes for a zinc finger protein. We propose that there is a reasonable probability that LD5-1 hybridizes with the gene for hereditary hemochromatosis.

Keywords: hybridization, cloning, yeast artificial chromosome.

Reprint requests to: Ernest Beutler, M.D., Department of Molecular and Experimental Medicine (SBR-3), The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, California USA, phone (619) 554-8040, fax (619) 554-6927, e-mail: beutler@scripps.edu.