Summary
The kinetics of lactate dehydrogenase in mouse cardiac muscle fibres, skeletal muscle fibres, gastric parietal cells, parotid gland ductal and acinar cells, oocytes and mouse and human hepatocytes were studied as a function of substrate concentration in sections of unfixed mouse and human tissues incubated at 37°C on lactate agarose gel films. The absorbances of the final reaction products deposited in single cells of various types were measured continuously as a function of incubation time using an image analysis system. The initial velocities (v i) of the dehydrogenase were calculated from two equations deduced previously by us, v i = a1∘A (equation 1) and v i = v + a 2∘A (equation 2), where v and ∘A are, respectively, the gradient (steady-state velocity) and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min, and a 1 and a 2 are constants characteristic for each cell type.
Hanes plots using v i, calculated from equation 2 gave more consistent estimates of the Michaelis constant (K m) and the maximum reaction velocity (V max ) than those employing either steady-state velocity measurements or v i calculated from equation 1. The K m thus found for mouse skeletal muscle fibres (10.4–12.5 mM) and hepatocytes (14.3–16.7 mM) agreed well with values determined previously in biochemical assays. However, the K m for cardiac muscle fibres (13.4 mM) was higher. The K m of the enzyme in gastric parietal cells, parotid gland cells and oocytes was in the range 7.6–9.7 mM. The Vmax were more diverse, ranging from 29 μmoles hydrogen equivalents/cm3 cytoplasm/min units in mouse parotid gland acinar cells, 59–68 units in skeletal and cardiac muscle fibres, 62–65 units in gastric parietal cells and oocytes, and 102–110 units in hepatocytes. The diversity found for K m and V max in different cell types confirms the value of the quantitative histochemical approach in revealing the heterogeneity of cellular metabolism in situ.
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References
Battellino, L. J. & Blanco, A. (1970) Catalytic properties of the lactate dehydrogenase isozyme ‘X’ from mouse testis. J. Exp. Zool. 174, 173–86.
Battellino, L. J., Jaime, F. R. & Blanco, A. (1968) Kinetic properties of rabbit testicular lactate dehydrogenase isozyme. J. Biol. Chem. 243, 5185–92.
Bennett, N. G. & Gutfreund, H. (1973) The kinetics of the interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamideadenine dinucleotide and lactate. Biochem. J. 135, 81–5.
Butcher, R. G. (1978) The measurement in tissue sections of the two formazans derived from Nitroblue Tetrazolium in dehydrogenase reactions. Histochem. J. 10, 739–44.
Costello, L. A. & Kaplan, N. O. (1963) Evidence for two forms of M-type lactate dehydrogenase in the mouse. Biochim. Biophys. Acta 73, 658–60.
Everse, J. & Kaplan, N. O. (1973) Lactate dehydrogenases: structure and function. In Advances in Enzymology. Vol. 37 (edited by Meister, A.), pp. 61–133. New York, London: John Wiley & Sons.
Gebhardt, R. (1992) Metabolic zonation of the liver: regulation and implications for liver function. Pharm. Ther. 53, 275–354.
Hawtrey, C. O., Naidu, A., Nelson, C. & Wolfe, D. (1975) The physiological significance of LDH-X. In Isozymes II. Physiological Function. (edited by Markert, C. L.), pp. 171–80. New York, London: Academic Press.
Jonges, G. N., VanNoorden, C. J. F. & Lamers, W. H. (1992) Insitu kinetic parameters of glucose-6-phosphatase in the rat liver lobulus. J. Biol. Chem. 267, 4878–81.
Jungermann, K. & Katz, N. (1989) Functional specialization of different hepatocyte populations. Physiol. Rev. 69, 708–64.
Junqueira, L. C., Carneiro, J. & Long, J. A. (1986) Basic Histology, 5th edn. Los Altos: Lange Medical Publications.
Katz, N. R. (1989) Methods for the study of liver cell heterogeneity. Histochem. J. 21, 517–29.
Markert, C. L. & Ursprung, H. (1962) The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Dev. Biol. 5, 363–81.
Nakae, Y. & Stoward, P. J. (1992) Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system. Histochemistry 98, 7–12.
Nakae, Y. & Stoward, P. J. (1993a) Estimating the initial reaction velocity of a soluble dehydrogenase in situ. Histochem. J. 25, 199–205.
Nakae, Y. & Stoward, P. J. (1993b) Kinetic analysis of lactate dehydrogenase in situ in mouse liver determined with a quantitative histochemical technique. Histochem. J. 25, 206–12.
Nakae, Y. & Stoward, P. J. (1994) The initial reaction velocities of lactate dehydrogenase in various cell types. Histochem. J. 26, 283–291.
Nisselbaum, J. S. & Bodansky, O. (1963) Purification, kinetic, and immunochemical studies of the major variants of lactic dehydrogenase from human liver, hepatoma, and erythrocytes; comparison with the major variant of human heart lactic dehydrogenase. J. Biol. Chem. 238, 969–74.
Nisselbaum, J. S., Packer, D. E. & Bodansky, O. (1964) Comparison of the actions of human brain, liver, and heart lactic dehydrogenase variants on nucleotide analogues and on substrate analogues in the absence and in the presence of oxalate and oxamate. J. Biol. Chem. 239, 2830–4.
Pagliaro, L. & Taylor, D. L. (1988) Aldolase exists in both the fluid and solid phases of cytoplasm. J. Cell Biol. 107, 981–91.
Pesce, A., Mckay, R. H., Stolzenbach, F., Cahn, R. D. & Kaplan, N. O. (1964) The comparative enzymology of lactic dehydrogenases. I. Properties of the crystalline beef and chicken enzymes. J. Biol. Chem. 239, 1753–61.
Pesce, A., Fondy, T. P., Stolzenbach, F., Castillo, F. & Kaplan, N. O. (1967) The comparative enzymology of lactic dehydrogenases. III. Properties of the H4 and M4 enzymes from a number of vertebrates. J. Biol. Chem. 242, 2151–67.
Schwert, G. W., Miller, B. R. & Peanasky, R. J. (1967) Lactic dehydrogenase. X. A re-evaluation of the effects of pH upon the kinetics of the reaction. J. Biol. Chem. 242, 3245–52.
Stoward, P. J. & Nakae, Y. (1988) Simultaneous histochemical assay of two dehydrogenases in the same cell. Histochem. J. 20, 610–6.
Südi, J. (1974) Macroscopic rate constants involved in the formation and interconversion of the two central enzyme-substrate complexes of the lactate dehydrogenase turnover. Biochem. J. 139, 261–71.
Swezey, R. R. & Epel, D. (1986) Regulation of glucose-6-phosphate dehydrogenase activity in sea urchin eggs by reversible association with cell structure elements. J. Cell Biol. 103, 1509–15.
VanNoorden, C. J. F., Kooij, A., Vogels, I. M. C. & Frederiks, W. M. (1985) On the nature of the ‘nothing dehydrogenase’ reaction. Histochem. J. 17, 1111–8.
Wachsmuth, E. D. (1980) Assessment of immunocytochemical techniques with particular reference to the mixed-aggregation immunocytochemical technique. In Trends in Enzyme Histochemistry and Cytochemistry (Ciba Foundation Symposium. 73) pp. 135–60. Amsterdam, Oxford: Excerpta Medica.
Wróblewski, F. & Gregory, K. F. (1961) Lactic dehydrogenase isozymes and their distribution in normal tissues and plasma and in disease states. Ann. N.Y. Acad. Sci. 94, 912–32.
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Nakae, Y., Stoward, P.J. The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell. Histochem J 26, 292–297 (1994). https://doi.org/10.1007/BF00157761
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DOI: https://doi.org/10.1007/BF00157761