PURIFICATION AND CHARACTERIZATION OF A CATALYTIC DOMAIN OF RAT INTESTINAL PHOSPHOLIPASE B LIPASE ASSOCIATED WITH BRUSH-BORDER MEMBRANES/

A brush border membrane-associated phospholipase B/lipase was solubili zed from the distal two-thirds of rat small intestine by autolysis dur ing storage at -35 degrees C over 1 month, and then the enzyme was pur ified to homogeneity and characterized enzymatically and structurally, The purified enzyme exhibited broad substrate specificity including e sterase, phospholipase A(2), lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chr omatographic analyses demonstrated that a single enzyme catalyzes thes e activities. It preferred hydrolysis at the sn-2 position of diacylph ospholipid and diacylglycerol without strict stereoselectivity, wherea s it apparently exhibited no positional specificity toward triacylglyc erol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases, inhibited purified enzyme, When the position o f enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a similar to 150-kD a protein; autolysis gave a 35-kDa product, in agreement with the resu lts of immunoblot analysis, The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH2-terminal a mino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of similar t o 38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F,, Ting, L., Urbain, T,, Komatsubara, T., Hatano, O., Okamoto, M,, and Tojo, H. (1998) J. Biol. Chem. 273, 2222 -2231). These results indicate that the purified enzyme is the catalyt ic domain derived from the second repeat of brush border membrane-asso ciated phospholipase B/lipase.