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Phospholipases A₁ and A₂ in Subcellular Fractions and Plasma Membranes of Krebs II Ascites Cells

Institut National de la Santé et de la Recherche Médicale, Unité de Recherches 101, Hópital Purpan, 31052 Toulouse, France

A method is described for the localization and characterization of phospholipases A₁ and A₂ (EC 3.1.1.4) in Krebs II ascites cells, particularly in the plasma membranes. Cells were lysed with a Dounce homogenizer in an isotonic sucrose medium. Plasma membranes sediment with mitochondria and lysosomes during subcellular fractionation and are finally isolated on a continuous sucrose gradient. The membranes are localized at two levels in the gradient, at densities of 1.06 and 1.15, in which 5'-nucleotidase (EC 3.1.3.5) activity exhibits a 9- and 21-fold purification, respectively. Total contamination by endoplasmic reticulum, lysosomes, and mitochondria is 17% for the low-density membrane fraction and 25% for the high-density fraction.

The phospholipases A present in Krebs II cells are active at pH 4.0 and pH 7.5. At the 2 pH values, they have A₁ and A₂ specificities. The intracellular distribution of acidic forms is comparable to that of acid phosphatase (EC 3.1.3.1), while neutral forms are localized like lactate dehydrogenase (EC 1.1.1.27).

A small proportion of neutral phospholipase A₂ has the same repartition on the sucrose gradient as nicotinamide adenine dinucleotide diaphorase (EC 1.6.4.3), an endoplasmic reticulum marker, and as 5'-nucleotidase, a plasma membrane marker.

Received 1/25/77. Accepted 8/30/77.