Extraction and Subcellular Localization of Porcine Lactate Dehydrogenase Activity and Isoenzymes

Abstract

The conditions for a total extraction of LDH activity and isoenzymes from pig heart, skeletal muscle, liver and spleen were described. Optimal extraction of LDH isoenzymes was obtained by the use of 0.1 M phosphate or tris buffers as extraction media and Ultra-Turrax homogenizer for destruction of tissues. Extraction of isoenzymes mainly composed by M subunits (LDH(5) and LDH(4)) was highly dependent on ionic strength of the homogenizing medium. Maximal release of LDH(2) and LDH(3) was found at a low ionic strength, whereas LDH(1) is independent of ion concentration of extraction medium. The release of cellular LDH isoenzymes by a perfusion of liver confirms the easy removal of LDH(1) and LDH(2) from cells and it is suggested that these isoenzymes exist as free enzymes in situ. Due to certain limitations of techniques available for investigation of the subcellular distribution of LDH isoenzymes, which makes it impossible to differentiate between a true binding of LDH isoenzymes to cell structures or an artificial adsorption to the surface of particles, it is implied that the question about a cellular compartmentalization of LDH isoenzymes still remains unclear. However from the present investigation it is clear that minimally 90 % of LDH is free in Cytoplasma and furthermore that if the remaining 10% is bound or adsorbed at all, the binding is of very loose character and may easily be abolished by the use of a suitable extraction medium.

Intracellular Distribution of Lactate Dehydrogenase Isoenzymes in Brain Tumours

“…Usually normal mitochondria displayed strong LDH 1 isoenzyme activity in all investigated tissues. This is generally explained by the oxidative metabolism of mitochondria [7,8], but other factors such as ionic strength of the homogenizing medium are also involved [9]. In our experiments of cellular fractions from brain tumours even mitochondria pattern changed from LDH 1 toward LDH 5.…”

Proton NMR observation of redox potential in liver

“…Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg body weight), and the liver was immediately removed from the animal. Approximately 1 g of liver tissue was homogenized in 5 volumes of 0.1 M phosphate buffer, and the homogenate was centrifuged at 18000g at 4 °C for 30 min (Hyldgaard-Jensen & Valenta, 1970). LDH activity in the diluted supernatant was then measured in the reaction mixture containing saturating concentrations of pyruvate (0.63 mM) and NADH (0.18 mM) as the substrates .…”