Assay for the enzymatic conversion of indoleacetic acid to 3-methylindole in a ruminal Lactobacillus species.
An assay to measure the rate of enzymatic formation of 3-methylindole (3MI) from indoleacetic acid (IAA) in Lactobacillus sp. strain 11201 was developed. The reaction mixture contained 50 micrograms of microbial protein per ml (range, 25 to 100 mg/ml), essential low-molecular-weight reaction ingredients, and radiolabeled IAA as substrate (range, 0 to 2 mM IAA). The reaction was anaerobic for 25 min at 39 degrees C. The apparent Michaelis-Menten constants were: Km, 0.14 mM IAA; and Vmax, 64 nmol 3MI.mg-1.min-1. The inhibitors avidin, aminopterin, and EDTA had no effect on the 3MI-forming enzyme. Dithionite stimulated the 3MI-forming enzyme. The product of the reaction, 3MI, acted as a noncompetitive inhibitor of the enzyme. Enzyme activity was associated with the cell wall fraction after sonication; treatment with the French press; or treatment with detergents, proteolytic enzymes, and EDTA.
Honeyfield
D C
DC
Department of Animal Sciences, Washington State University, Pullman 99164-6320.
Carlson
J R
JR
D000818:Animals; D002417:Cattle; D004798:Enzymes / Q000378:metabolism; D007210:Indoleacetic Acids / Q000378:metabolism; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D007778:Lactobacillus / Q000378:metabolism*; D012417:Rumen / Q000382:microbiology*; D012862:Skatole / Q000378:metabolism*
D004798:Enzymes; D007210:Indoleacetic Acids; D007211:Indoles; C030737:indoleacetic acid; D012862:Skatole