Histidine decarboxylase measurement in brain by 14CO2 trapping.
A method for measuring histidine decarboxylase (HDC) in crude rat brain homogenates was developed by modification of existing 14CO2-trapping methods. The addition of EDTA to tissue homogenates and assay buffer reduced non-enzymatic decarboxylation, and improved assay sensitivity and reliability. Addition of polyethylene glycol (molecular weight 300, PEG300) to the homogenizing buffer increased enzyme stability, permitting storage of crude homogenates. Studies of time course, tissue dilution and blanks showed that up to 8 mg of tissue could be assayed successfully with a 3.5-hr incubation. S-alpha-Fluoromethylhistidine (FMH) and alpha-hydrazinohistidine, specific inhibitors of HDC, induced concentration-dependent reductions of enzyme activity by up to 90%, whereas inhibitors of other decarboxylases had little or no effect. Kinetic studies of the enzyme in crude homogenates yielded Km and Vmax values similar to those found previously with other HDC methods, although a poor fit was found to a single enzyme model. When determined by the new method, the distribution of HDC in seven regions of the rat brain agreed well with previous results. The method is rapid, simple to perform, and requires no specialized equipment other than a scintillation counter.
Kishikawa
H
H
Department of Pharmacology and Toxicology, Albany Medical College, NY 12208.
Willinger
S M
SM
Jackowski
S
S
Barke
K
K
Hough
L B
LB
D000818:Animals; D001923:Brain Chemistry; D002245:Carbon Dioxide / Q000032:analysis*; D002250:Carbon Radioisotopes; D006639:Histidine / Q000031:analogs & derivatives / Q000494:pharmacology; D006640:Histidine Decarboxylase / Q000032:analysis* / Q000037:antagonists & inhibitors; D007700:Kinetics; D008297:Male; D008762:Methylhistidines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012588:Scintillation Counting
D002250:Carbon Radioisotopes; D008762:Methylhistidines; D002245:Carbon Dioxide; C045518:alpha-hydrazinohistidine; D006639:Histidine; C028309:alpha-fluoromethylhistidine; D006640:Histidine Decarboxylase