Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane.
A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.
Gassama-Diagne
A
A
Institut National de la Santé et de la Recherche Médicale Unité 101, Biochimie des Lipides, Toulouse, France.
Fauvel
J
J
Chap
H
H
D000818:Animals; D002118:Calcium / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000201:enzymology*; D008245:Lysophospholipase / Q000302:isolation & purification*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity
D010743:Phospholipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase; D002118:Calcium