Antibodies to a synthetic peptide corresponding to a Ser-40-containing segment of tyrosine hydroxylase: activation and immunohistochemical localization of tyrosine hydroxylase.
A peptide corresponding to position 32-47 in tyrosine hydroxylase was synthesized (TH-16) and polyclonal antibodies against this peptide were raised in rabbits (anti-TH-16). The effects of anti-TH-16 on modulation of tyrosine hydroxylase activity were investigated. Anti-TH-16 enhanced the enzymatic activity in a concentration-dependent manner, and the antigen TH-16 inhibited the stimulatory activity of the antiserum in a concentration-dependent manner. The activated enzyme had a lower Km app for the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and a higher Vmax app than the nonactivated enzyme. Anti-TH-16 was characterized further by its ability to immunoprecipitate the enzyme activity by labeling tyrosine hydroxylase after Western blotting and by immunohistochemical labeling of catecholaminergic neurons. Anti-TH-16 did not block activation of tyrosine hydroxylase by phosphorylation catalyzed by cyclic AMP-dependent protein kinase. Exposure of the enzyme to anti-TH-16 and subsequent phosphorylation of the enzyme resulted in a greater activation of the enzyme than the sum of activation produced by these two treatments separately. However, the activation was less than additive when the enzyme was first phosphorylated and subsequently exposed to anti-TH-16. The present study demonstrates the utility of anti-TH-16 in investigating the molecular aspects of the enzyme activation.
Lee
K Y
KY
Neurochemistry Research Laboratory, New York University Medical Center, New York 10016.
Lew
J Y
JY
Tang
D
D
Schlesinger
D H
DH
Deutch
A Y
AY
Goldstein
M
M
D000310:Adrenal Gland Neoplasms / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex / Q000032:analysis; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D010446:Peptide Fragments / Q000276:immunology; D010673:Pheochromocytoma / Q000201:enzymology; D010766:Phosphorylation; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D010446:Peptide Fragments; D014446:Tyrosine 3-Monooxygenase