Purification of lysophospholipase and lysophospholipase-transacylase from rabbit myocardium

This chapter describes the chromatographic methods developed for the purification of rabbit myocardial lysophospholipase and lysophospholipase-transacylase to homogeneity. Lysophospholipase and lysophospholipase-transacylase activities are quantified by incubating 1-[1-¹⁴C]hexadecanoyl-sn-glycero-3-phosphocholine (palmitoyllysophosphatidylcholine) with 50 or 100 μl of column fractions in 75 mM potassium phosphate, pH 7.0 for 10 min at 37 ° in a final volume of 700 μl. New Zealand White rabbits (typically 10–20) are sacrificed by cervical dislocation, a left thoracotomy is performed, and hearts (∼6 g/heart) are rapidly placed in ice-cold buffer. All procedures are performed at 0°–4°. Ventricular myocardium is isolated, extensively rinsed in buffer, and homogenized utilizing three 30 sec bursts from a Polytron homogenizer at a setting of 5 to yield a 25% homogenate. Nuclei, mitochondria, and cellular debris are removed by low-speed centrifugation, and the resulting supernatant is further centrifuged at 100,000 g_max for one hour to obtain myocardial cytosol. Myocardial cytosol is brought to 55% saturation by slow addition of solid ammonium sulfate, the mixture is stirred at 4° for an additional nine min, and the resulting precipitate is pelleted by centrifugation at 10,000 g_max for 5 min.