A rapid, sensitive assay for glycine-directed amidating enzymes.
Current assays for glycine-directed, peptide-amidating enzymes have several shortcomings. In this report, we describe a rapid, sensitive microassay for amidating activity which overcomes these disadvantages. Tissue homogenates are incubated in the presence of D-Tyr-Val-Gly-OH and the product, D-Tyr-Val-NH2, is measured by radioimmunoassay using an antiserum with an affinity for the product, D-Tyr-Val-NH2 (Kaff 2 X 10(8) L/M), 4 orders of magnitude higher than for the substrate, D-Tyr-Val-Gly-OH (Kaff 4 X 10(4) L/M), and 3 orders of magnitude higher than for the deamidated product D-Tyr-Val-OH (Kaff 8 X 10(5) L/M). Addition of N-ethylmaleimide (0.5 mM) to the enzyme incubates prevents the degradation of D-Tyr-Val-NH2 and permits the measurement of enzymatic activity in crude homogenates. This assay is sensitive enough to permit the measurement of amidating activity in crude rat brain homogenates containing as little as 4-8 micrograms protein.
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000201:enzymology*; D004151:Dipeptides / Q000032:analysis; D008722:Methods; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology*; D011863:Radioimmunoassay; D051381:Rats; D013552:Swine
D004151:Dipeptides; D009097:Multienzyme Complexes; C047208:tyrosylvalinamide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors