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Purification and characterization of a catalytic domain of rat intestinal phospholipase B/lipase associated with brush border membranes.

Authors: Tojo, H  Ichida, T  Okamoto, M 
Citation: Tojo H, etal., J Biol Chem. 1998 Jan 23;273(4):2214-21. doi: 10.1074/jbc.273.4.2214.
Pubmed: (View Article at PubMed) PMID:9442064
DOI: Full-text: DOI:10.1074/jbc.273.4.2214

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.

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CRRD Object Information
CRRD ID: 30309923
Created: 2020-06-20
Species: All species
Last Modified: 2020-06-20
Status: ACTIVE



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RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.