Micromethods in single muscle fibers. 1. Determination of catalase and superoxide dismutase.
Methods have been developed for the measurements of catalase and superoxide dismutase (SOD) in single, isolated muscle fibers. These fibers are also classified according to fiber type. Catalase is determined using a fluorescent method for the measurement of hydrogen peroxide consumed. SOD measurements are carried out using a modification of established techniques whereby the inhibition of oxidation of epinephrine by SOD is assayed fluorometrically. Both enzymes may be determined in submicrogram samples of dried muscle. This approach avoids the complication of the inclusion of nonmuscle tissue with varying enzymatic activities which is frequently experienced when using homogenates of muscle, particularly diseased muscle. In addition, these techniques can be used to determine the inherent variation in SOD and catalase activities within individual fibers of the same fiber type. The Km and Vmax for catalase, determined using homogenates of human muscle, were found to be 12 mM and 1.45 mumol/min/mg dry wt, respectively. Catalase of muscle was inhibited 50% by 2 microM sodium azide. Mn-SOD contributes less than one-fifth of the total SOD activity. Therefore the activity is largely due to the Cu-Zn form of SOD. These methods are applicable to a wide variety of tissues.
Austin
L
L
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Arthur
H
H
de Niese
M
M
Gurusinghe
A
A
Baker
M S
MS
D002374:Catalase / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D009132:Muscles / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000032:analysis*
D002374:Catalase; D013482:Superoxide Dismutase