Publications

• Purification of the lysosomal acid lipase from human liver and its role in lysosomal lipid hydrolysis.

  Warner T.G., Dambach L.M., Shin J.H., O'Brien J.S.

  The lysosomal acid lipase has been purified 2,500-fold to near homogeneity from human liver. The enzyme was converted to a soluble form by extraction of frozen tissue with Triton X-100. The enzyme, which required Triton X-100 in buffers at all purification steps for optimal yields, was stabilized ... >> More

  The lysosomal acid lipase has been purified 2,500-fold to near homogeneity from human liver. The enzyme was converted to a soluble form by extraction of frozen tissue with Triton X-100. The enzyme, which required Triton X-100 in buffers at all purification steps for optimal yields, was stabilized by the inclusion of 33% ethylene glycol during purification. Lectin chromatography on concanavalin A-Sepharose followed by chromatography on carboxymethyl-cellulose and Sephadex G-150 provided the highly purified enzyme in 17% yield. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the minimum molecular weight was about 29,000 +/-1,000. Minor protein contaminants at Mr = 58,500, 14,700 and 13,900 were present in the final preparation. A single protein band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Gel filtration on Sephadex G-150 in the presence of Triton X-100 gave an apparent molecular weight of about 125,000 +/-13,000. Trioleoylglycerol, cholesterol oleate, and 1,2- and 1,3-dioleoylglycerols were substrates for the purified enzyme giving apparent Vmax values of 5,400, 1,400, 19,400, and 22,100 nmol min-1 mg of protein-1, respectively, and Km values of 0.8, 0.8, 0.9, and 1.2 mM, respectively. The recoveries of both trioleoylglycerol and cholesterol oleate hydrolytic activities were nearly identical at each purification step, suggesting that the acid lipase as single enzyme is responsible for lysosomal hydrolysis of the neutral lipids. Monooleoylglycerols were not substrates for the enzyme. << Less

  J. Biol. Chem. 256:2952-2957(1981) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Messenger RNAs expressed in intestine of adult but not baby rabbits. Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity.

  Boll W., Schmid-Chanda T., Semenza G., Mantei N.

  Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S- ... >> More

  Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins. One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus. The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli. The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes. Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity. << Less

  J. Biol. Chem. 268:12901-12911(1993) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.