Activating antibodies to tyrosine hydroxylase.
Crude immunoglobulin fractions from a sheep, immunized with rat pheochromocytoma tyrosine hydroxylase, increased the catalytic activity of tyrosine hydroxylase in vitro up to 20-fold. Kinetic analysis of the activation revealed both a decrease in Km(app) for pterin cofactor and an increase in Vmax(app). No change Km(app) for tyrosine was observed. Activation was maximal at the minimum antibody/enzyme ratio required to remove tyrosine hydroxylase activity from solution with protein A immunoadsorbent. Chromatography of the crude immunoglobulins on either DEAE-Sephacel or protein A-Sepharose separated two immunoglobulin fractions. In both cases, the activating influence and the anti-tyrosine hydroxylase activity bound to the matrices and were eluted together. As supported by data from a solid phase radioimmunoassay using rabbit anti-bovine IgG1, this chromatographic data was consistent with the activating activity and the anti-tyrosine hydroxylase activity both being sheep IgG1 immunoglobulins. Preadsorption of the active immunoglobulin fraction from DEAE-Sephacel chromatography (with protein A immunoadsorbent precoated with rabbit anti-sheep IgG) eliminated the activating influence. Moreover, after incubation with the active fraction, tyrosine hydroxylase was bound to protein A-Sepharose and the activity recovered was severalfold higher than the initial activity. These data clearly demonstrate the involvement of antibody in the activation of the enzyme and suggest that the anti-tyrosine hydroxylase activity and the activating activity may derive from one and the same set of interactions.
D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D007163:Immunosorbent Techniques; D009374:Neoplasms, Experimental / Q000201:enzymology; D010673:Pheochromocytoma / Q000201:enzymology; D051381:Rats; D013205:Staphylococcal Protein A / Q000276:immunology; D013211:Staphylococcus aureus; D014446:Tyrosine 3-Monooxygenase / Q000276:immunology* / Q000378:metabolism
D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D014446:Tyrosine 3-Monooxygenase