Abstract
A monoacylglycerol-hydrolyzing enzyme has been purified 2500-fold from rat adipose tissue. The key step was the solubilization of the enzyme, presumably as an enzyme-detergent complex, by sonication with a nonionic polyoxyethylene alcohol detergent. The purification was achieved by ion exchange and gel chromatography, and isoelectric focusing, in the presence of detergent. By sodium dodecyl sulfate gel electrophoresis the enzyme protein was more than 85% pure. This method indicated a minimum molecular weight of 32,900. The preliminary amino acid composition, excluding tryptophan, could best be fitted with a value of 31,800. The purified enzyme had a pI of 7.2, an estimated Stokes radius of 39 A by gel chromatography and a pH optimum of 8.0. Enzyme stability was highly dependent on presence of detergent and free sulfhydryl groups. The enzyme was responsible for the main monoacylglycerol- but only a small part of the p-nitrophenylacetate-hydrolyzing activity of crude adipose tissue extracts and hydrolyzed 1(3)- and 2-monooleoylglycerol at equal rates. Under the assay conditions used it did not catalyze the hydrolysis of emulsified trioleoylglycerol, micellar or emulsified dioleoylglycerol, emulsified cholesterol oleate or micellar lysophosphatidylcholine. It is possible that the enzyme may be a specific monoacylglycerol hydrolase.
Highlights
There was no indication of any other enzymatic activity against monoacylglycerols in these extracts
Before solubilization with detergent the enzymatic activity was found in particles or aggregates of large molecular size containing a high proportion of phospholipids, cholesterol, and some triacylglycerol and was precipitated together with considerably more acidic proteins at pH 5.2
The monoglyceridase activity of the purified hormone-sensitive lipase preparation of Heller and Steinberg [5] may be due to the same enzyme, since pH optimum and temperature inactivation data are similar and both purifications start from the same tissue preparation
Summary
The nonionic detergent Nonipol TD 12, a polydisperse preparation of tridecyl polyethoxyethanols with a calculated average molecular weight of 728, was delivered by Rexoline Chemicals, Helsingborg, Sweden It had a cloud point of 93” measured in a 19 aqueous solution and a calculated hydrophile-lipophile balance of 14.5 [9]. Lysophospholipase activity was assayed at ~21” for 2 hours using 1 rnM [3H]oleic acid labeled llysophosphatidylcholine in 0.1 M ‘Iris-HCI, pH 8.0, with or without. One unit of enzyme activity in all cases corresponds to the release of 1 wmol of fatty acid/min at 21’ and specific activity is defined as units of’ enzymatic activity per mg of protein, Isoelectric Focusing-Isoelectric focusing was performed with the LKB X100-10 electrofocusing column (1:)) with a 0 to 50% (w/v) sucrose gradient containing 1’; (w/v) Ampholine, 3 rnM Nonipol,. Amino acid analyses were performed with a Durrum D-500 analyzers using the single column technique described by the manufacturer
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