[A microphotometric study of the kinetics of the histochemical reaction to lactate dehydrogenase].
The aim of the work--to ascertain and substantiate concentrations of the main components of the incubation medium used for identification and quantitative evaluation of the activity of lactate dehydrogenase in cryostat sections. Studies were conducted using cryostat sections of the rat's liver as thick as 5, 10, 15 mcM with concentration in the incubation medium of lactate 25--125 mM, NAD 2--12 mM, nitrobleu and tetranitrobleu tetrazoles 0.3--5 mM, and also under conditions with addition of pyruvate in concentrations from 0.3 to 100 mM. The cytospectrophotometric method was used for evaluation of kinetic parameters of the histochemical reaction. The data on the dependence of the rate of tetrazole reduction upon the concentration of substrate, cofactor, thickness of the section and the level of the end product of the pyruvate reaction in the incubation medium were obtained. The main kinetic parameters of the enzymatic reaction in liver sections--value of Michaelis' constant and the concentration of the substrate at which the maximal rate of the reaction was observed--were calculated. Recommendations for selection of an optimal composition of the incubation medium, ensuring the strictly quantitative evaluation of the activity of an enzyme in cryostate sections are suggested.
D000818:Animals; D006651:Histocytochemistry / Q000379:methods*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D051381:Rats; D013053:Spectrophotometry / Q000379:methods
D007770:L-Lactate Dehydrogenase
Mikrofotometricheskoe issledovanie kinetiki gistokhimicheskoĭ reaktsii na laktatdegidrogenazy