This project focuses on the specific enzymology and cell-biology underlying the burst of superoxide and hydrogen peroxide production that occurs when certain phagocytic leukocytes are stimulated. The relevant cells are granulocytes, and elicited or immunologically activated [but not resident (control)] peritoneal macrophages. The same cells from different species may employ different enzymes or enzyme systems for production of active oxygen species. Further, each cell type may produce 02- and H202 through the action of more than one enzyme or enzyme system. These comparative aspects will be studied. The role of the cell-membrane, to which the enzymes and enzyme systems mentioned are linked in varying degrees, will be tested. This applies both to effects of membrane lipid components per se on the function (kinetics, specificity, etc.) of the enzymes, as well as to aspects of the physical nature of the membrane, e.g., relevance of lipid domains. These matters will be explored by incorporating purified enzymes into liposomes of known lipid composition, and at the whole cell level, by probing the membrane with lipid substances, particularly unsaturated fatty acids, that trigger the respiratory burst. Physical measurements of the state of the membrane under such conditions are also planned. The question will be explored as to whether enzyme function may be changed by structural modification of enzymes when the cells are stimulated, e.g. conversion of NAD(P)+ linked dehydrogenases to oxidases by proteolysis or oxidation of sulfhydryl groups. Functions of a b-like cytochrome suspected of being involved in the respiratory burst may also be affected by the former modification. A crucial matter is the definition of the """"""""trigger"""""""" that activates the enzyme systems - probably related to physical alteration of the membrane milieu or chemical modification of the enzymes, as indicated above. The cellular locale(s) of the enzyme(s) that produce O2- and H2O2 will be explored both by cytochemical techniques involving capture of the product(s), and by immunological methods. The latter should allow localization of the enzyme(s) even when the cells are in an unstimulated state. The main objective of these studies is to deepen insight into the ways the O2- H2O2 producing system(s) provide(s) antibacterial and antitumor potential to these cells, by obtaining information on the nature of the enzymes involved, their cytological locale(s), and their regulation at the cellular level.
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