Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase.
Cholesterol ester hydrolase (sterol-ester acylhydrolase, EC 3.1.1.13) was purified from human pancreatic tissue by column chromatography and acetone precipitation, leading to a 400-fold enrichment. Isoelectric focusing of this product reveals a double-band at pH 4.5 and 4.6. The molecular weight was estimated at 320 kDa by means of Sephadex filtration on calibrated columns. Obviously these large molecules represent a tetrameric form of the monomeric subunit (molecular mass 76-80 kDa), which is also enzymatically active. It was found together with the dimeric form in pancreatic juice, where the tetrameric enzyme is responsible for the major part of the hydrolytic activity, splitting cholesterol ester as well as synthetic substrates, such as fluorescein or p-nitrophenyl esters. Attempts to split the tetrameric cholesterol ester hydrolase, isolated from pancreatic tissue, into active subunits found additionally in pancreatic juice by the influence of bile acids and proteolytic enzymes failed. The spectral shift method using Rhodamine fluorescence was employed in order to prove that fluorescein dilaurate forms micellar solutions and mixed micelles when bile salts are present.
Meyer
J G
JG
Zentrallaboratorium, Stadtkrankenhaus Hanau, Universität Frankfurt, F.R.G.
D000096:Acetone; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008066:Lipolysis; D046911:Macromolecular Substances; D008823:Micelles; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010189:Pancreatic Juice / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence; D002787:Sterol Esterase / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013656:Taurocholic Acid / Q000494:pharmacology
D046911:Macromolecular Substances; D008823:Micelles; D000096:Acetone; D013656:Taurocholic Acid; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase; D010447:Peptide Hydrolases