Kinetic parameters of lactate dehydrogenase in liver and gastrocnemius determined by three quantitative histochemical methods.
We determined the Michaelis constant (K(m)) and maximal velocity (Vmax) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (vi) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The Km and Vmax were calculated from Hanes plots of s/vi on L-lactate concentration (s). The Km values obtained with three assay methods were similar and in the range of 21.1-21.9 mM for pure LDH, 8.62-13.5 mM for LDH in mouse periportal hepatocytes, and 13.3-17.9 mM for LDH in mouse skeletal muscle fibers. The Vmax values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity kcat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K(m) values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.
Nakae
Y
Y
Department of Oral Anatomy 1, Tokushima University School of Dentistry, Japan.
Stoward
P J
PJ
D000818:Animals; D007091:Image Processing, Computer-Assisted; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology*; D011142:Polyvinyl Alcohol / Q000494:pharmacology; D011817:Rabbits; D012685:Sepharose / Q000494:pharmacology; D013997:Time Factors; D014021:Tissue Preservation
D011142:Polyvinyl Alcohol; D012685:Sepharose; D007770:L-Lactate Dehydrogenase