The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell

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 degrees 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 (vi) of the dehydrogenase were calculated from two equations deduced previously by us, vi = a1 zero A (equation 1) and vi = v + a2 zero A (equation 2), where v and zero 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 a1 and a2 are constants characteristic for each cell type. Hanes plots using vi calculated from equation 2 gave more consistent estimates of the Michaelis constant (Km) and the maximum reaction velocity (Vmax) than those employing either steady-state velocity measurements or vi calculated from equation 1. The Km 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 Km for cardiac muscle fibres (13.4 mM) was higher. The Km of the enzyme in gastric parietal cells, parotid gland cells and oocytes was in the range 7.6-9.7 mM.