This study investigates the occurrence, physiological function, and enzymology of the sulfation of tyrosine residues in human plasma proteins. Three human plasma proteins-- the fourth component of complement (C4), heparin cofactor II, and alpha2-antiplasmin-- which contain tyrosine sulfate residues will be analyzed in detail. Objectives are: 1) To identify the sites and stoichiometry of sulfation of these proteins, 2) To examine the effects of sulfation on activity and stability of these proteins, 3) To characterize the process of sulfation and the sulfotransferase which mediates it, 4) To investigate the functions of the sulfate-containing domains in these proteins, 5) To determine whether there is physiological or pathological variation in the sulfation of proteins. Peptides containing the sites of sulfation of these proteins have been prepared by chemical synthesis and by cleavage of the purified proteins with cyanogen bromide. These peptides will be used for structural studies, generation of antisera specific for sulfated segments of the proteins, and examination of the function of sulfate-containing domains. The effects of sulfation on the function of C4, heparin cofactor II, and alpha2-antiplasmin will be examined directly by comparing the activity and stability of sulfated and nonsulfated forms of these proteins. Nonsulfated forms of protein will be obtained by incubating HepG2 cells with sulfation inhibitors or by incubating the proteins with arylsulfatase. HepG2 cells will serve as a model system for investigating the sulfation of these proteins and the influence of sulfate concentration, hormones, and drugs on this process. Enzymology of the sulfation of proteins will be analyzed by purification of the sulfotransferase from rat liver. Synthetic peptides corresponding to sites of sulfation will be used as ligands for affinity purification of the enzyme and as substrates for assaying its activity. Finally, this study will examine whether there is physiological or pathological variation in the sulfation of proteins. Plasma specimens will be analyzed by electrophoresis to measure the relative quantities of sulfated and nonsulfated forms of specific plasma proteins.
