Biospecific dye-binding and hydrophobic interaction chromatography as tools for high yield preparation of purified phospholipase A1 from rat liver.
A basically new approach is presented for purifying lysosomal phospholipase A1 (EC 3.1.1.32) from rat liver. This procedure not only simplifies and speeds up the purification process, but also improves the yield in comparison to the most efficient methods reported so far. A high recovery of about 88% was achieved by (1) homogenisation of whole rat liver in a hypotonic medium, (2) acid precipitation, (3) combined dye binding chromatography on triacinyl dyes (Yellow H-A and Red HE-3B) immobilised to agaroses, and (4) combined concanavalin A-Sepharose and phenyl-Sepharose chromatography. Ethylene glycol was required for enzyme stabilisation as well as for enzyme elution in dye-binding and hydrophobic chromatography. In SDS-polyacrylamide slab gel electrophoresis, the purified material showed two major protein bands of 56 and 33 kDa, which amounted to about 85 and 12%, respectively, of the total protein visualised. Under reductive conditions, the 56 kDa protein decomposed completely into three subunits of 30, 21 and 20 kDa. The 33 kDa protein in the non-reduced material seems to be identical with the 30 kDa protein in the reduced material. Native polyacrylamide gel electrophoresis provided strong evidence that the 56 kDa protein is the active form of PLA1. The purified material displayed a specific activity of approximately 7.7 mumol fatty acid released per min per mg of protein using 200 microM phosphatidylethanolamine as a substrate.
Löffler
B M
BM
Max-Planck-Institut für Experimentelle Medizin, Göttingen, F.R.G.
Kunze
H
H
D000818:Animals; D002846:Chromatography, Affinity; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification*; D054466:Phospholipases A1; D051381:Rats; D012995:Solubility
D003208:Concanavalin A; D010740:Phospholipases; D010741:Phospholipases A; D054466:Phospholipases A1