Immobilization of tryptophan hydroxylase by immune adsorption: a method to study regulation of catalytic activity.
Tryptophan hydroxylase (TPH) can be immobilized by adsorption to Pansorbin after binding to the monoclonal antibody PH8. This method yields recoveries of 35%-40% of total TPH activity in crude extracts and can be completed in 1.5 h. The immobilized form of TPH retains the essential kinetic properties of the native enzyme and responds to activators (phosphatidylserine) and inhibitors (catechol compounds) as does the native enzyme. Unlike TPH in brain extracts, immobilized TPH is not activated by calcium-stimulated phosphorylating conditions. When extracts from which TPH has been precipitated, and which contain calcium-calmodulin dependent protein kinase are added to immobilized TPH, the activation of TPH is restored. This method of immobilization of TPH via immune-adsorption allows for the highly specific and rapid preparation of affinity purified TPH that can be used to study the regulation of this enzyme by a variety of effectors, especially protein kinases.
Johansen
P A
PA
Department of Psychiatry, Wayne State University, School of Medicine, Detroit, MI 48207.
Jennings
I
I
Cotton
R G
RG
Kuhn
D M
DM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007163:Immunosorbent Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014365:Tryptophan Hydroxylase / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D010718:Phosphatidylserines; D000255:Adenosine Triphosphate; D014365:Tryptophan Hydroxylase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008274:Magnesium; D002118:Calcium