Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry
Year: 1985
Type: article
Abstract: Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.
Source: European Journal of Biochemistry
Authors: Jean-Dominique MONET, Mona BRAMI, Nicole DAUTIGNY, Jacques ASSAILLY, Marie BACHELET, Andree ULMANN, Cyrille A. BADER
Institutions: Hôpital Necker-Enfants Malades, Inserm
Language: English
Subfield: Rheumatology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2018917476