ARTICLE

Kinetic Parameters of Lactate Dehydrogenase in Liver and Gastrocnemius Determined by Three Quantitative Histochemical Methods

Correspondence to: Yoshiko Nakae, Dept. of Oral Anatomy 1, Tokushima Univ. School of Dentistry, 3 Kuramoto-cho, Tokushima 770, Japan.

We determined the Michaelis constant (K_m) and maximal velocity (V_max) 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 (v_i) of LDH, corrected for "nothing dehydrogenase," were determined as described in the previous article. The K_m and V_max were calculated from Hanes plots of s/vi on L-lactate concentration (s). The K_m 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 V_max values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreeement but were 53-65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity k_cat 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. (J Histochem Cytochem 45:1427-1431, 1997)

Key Words: lactate dehydrogenase, enzyme kinetics, quantitative histochemistry, image analysis, skeletal muscle, liver, michaelis constants, maximal reaction velocities

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This article has been cited by other articles:

				Y. Nakae and P. J. Stoward Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells J. Histochem. Cytochem., October 1, 1997; 45(10): 1417 - 1426. [Abstract] [Full Text] [PDF]

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