A brush border membrane-associated
phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by
autolysis during storage at -35 degrees C over 1 month, and then the
enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified
enzyme exhibited broad substrate specificity including
esterase,
phospholipase A2,
lysophospholipase, and
lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single
enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and
diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward
triacylglycerol. Diisopropyl
fluorophosphate, an irreversible inhibitor of
serine esterases and lipases inhibited purified
enzyme. When the position of
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 approximately 150-kDa
protein;
autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa
enzyme consisted of a 14-kDa
peptide and a glycosylated 21-kDa
peptide. Their NH2-terminal amino acid sequences were determined and found in the second repeat of 161-kDa
phospholipase B/lipase with 4-fold tandem repeats of approximately 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. (1988) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified
enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated
phospholipase B/lipase.