Analysis of Tween 80 as an esterase/ lipase substrate for lipolytic activity assay

The Tween 80 assay to detect lipolytic activities in agar media was evaluated. A spectrophotometric assay for Tween 80 hydrolysis was established. The specific activities with Tween 80, as well as with some conventional lipase-type and esterase-type substrates, were measured using several lipases and esterases. The activity with Tween 80 was similar to that obtained with p-nitrophenyl butyrate; the enzyme activities with both substrates were between the esterase and lipase categories. © Rapid Science Ltd. 1998     

A thermophilic, lipolytic Bacillus sp., and continuous assay of its p-nitrophenyl-palmitate esterase activity

Abstract The newly-isolated extremely thermophilic Bacillus sp. strain Wai28A5, able to grow at 70°C on tripalmitin and other triglycerides, possessed a p - nitrophenyl-palmitate esterase activity with a half-life of 60 min at 70°C and 12 min at 85°C. This activity was produced during exponential growth on tripalmitin, and the level of activity decreased once growth stopped. Transfer to tripalmitin-containing medium resulted in induction of the esterase activity. The activity was largely cell-associated (60 to 87% of the total activity). The p -nitrophenyl-palmitate esterase activity was proportional to the amount of culture added to enzyme assays and was destroyed by autoclaving, showing it to be enzymatic. A continuous assay for esterase activity was developed, and proved to be sensitive enough to detect 0.02 mU ml⁻¹ esterase activity. Maximal esterase activity was at 400 μM p -nitrophenyl-palmitate and the optimum pH (at 70°C) was 8.7.     

Isoenzymes of lipolytic acyl hydrolase and esterase in potato tuber

Five varieties of potato (Solanum tuberosum) were shown by gel- and free-flow-electrophoresis to exhibit multiple forms of lipolytic acyl hydrolase (LAH) and esterase enzymes. The electrophoretic patterns of LAH and esterase activities and protein differed with the variety and were characteristic for a given variety. In the variety (Golden Wonder) with the highest LAH activity (p-nitrophenylpalmitate as substrate), this was 200-fold greater than the esterase activity (p-nitrophenylacetate as substrate) and isoenzyme patterns for both enzymes were the most complex. In the variety with a very low LAH activity (Désirée), the LAH and esterase activities were similar and more simple isoenzyme patterns for these enzymes were observed.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.     

Characteristics of the protein kinase activity associated with rat neurofilament preparations

Some properties of the protein kinase activity associated with neurofilaments isolated from the brain stem and spinal cord of rats have been investigated. The activity had an apparent Km for ATP of 20 microM, a pH optimum of 8.0 and phosphorylated both serine and threonine residues in neurofilament proteins. Cyclic AMP had no effect on the in vitro reaction and casein was a preferred exogenous substrate in comparison to histone. Phosphopeptide mapping of the 145 kDa subunit from neurofilaments phosphorylated in the presence and absence of microtubule proteins indicated that the neurofilament-associated activity was distinct from the microtubule-associated protein kinase. Limited proteolysis of neurofilaments with chymotrypsin indicated that the enzyme activity was not associated with a domain of the 200 kDa subunit which may form the side-arm projections on neurofilaments.     

Isolation and characterization of four lipolytic preparations from rat skeletal muscle

Four different lipolytic preparations have been isolated from rat skeletal muscle. Two of these, provisionally designated monopalmitin lipase (MPL) and monomyristin lipase (MML), are associated with insoluble cellular particulate fractions. The other two enzymes, provisionally designated tricaproin lipase (TCL) and monolaurin lipase (MLL), are found in the high-speed supernatant fraction. Taken as a group, these enzymes are capable of hydrolyzing short-chain triglycerides (acyl moieties of C(3) to C(8)) and monoglycerides of lauric, myristic, palmitic, stearic, and oleic acids. All of these enzymes have a serine residue at or near the catalytic site as they are strongly inhibited by diisopropyl fluorophosphate. The two particulate preparations contain a sulfhydryl group and are sensitive to p-chloromercuribenzoate and N-ethylmaleimide, while the soluble preparations are not. The MLL, MML, and MPL preparations all have alkaline pH optima, while the TCL preparation has an acidic optimum. Buffer type is important: some buffer compounds completely inhibited one or more preparations. Of the soluble enzymes, MLL withstood heating to 60 degrees C, while TCL is completely inactivated at this temperature. Of the particulate preparations, only MML was stable to lyophilization. It is concluded that there are at least four lipolytic enzymes in rat skeletal muscle. The possible significance of the presence of these enzymes in muscle is discussed.     

Characterisation of steryl esterase activities in commercial lipase preparations

Triglycerides, steryl esters, resin acids, free fatty acids and sterols are lipophilic extractives of wood (commonly referred to as pitch or wood resin) and have a negative impact on paper machine runnability and quality of paper. Thus, enzymes capable of modifying these compounds would be potential tools for reducing pitch problems during paper manufacture. In this work, 19 commercial lipase preparations were tested for their ability to degrade steryl esters, which may play a significant role in the formation and stabilisation of pitch particles. Six lipase preparations were shown to be able to degrade steryl esters. Lipase preparations of Pseudomonas sp., Chromobacterium viscosum and Candida rugosa were shown to have the highest steryl esterase activities. The enzymes were able to hydrolyse steryl esters totally in the presence of a surfactant (Thesit). Up to 80% of the steryl esters were degraded in aqueous dispersion. Preliminary characterisation of the enzymatic activities revealed that the lipase preparation of Pseudomonas sp. could be the most potential enzyme in industrial applications. The steryl esterase activity of this preparation was stable over a broad pH range and the enzyme was able to act efficiently at pH 6-10 and at temperatures up to 70 degrees C.     

Extracellular lipolytic activity in Phoma glomerata

Several properties of the lipolytic activity exhibited by the conidial fungus Phoma glomerata were studied. Lipolytic activity in an aqueous buffer medium was measured on triacylglycerol, phosphoglyceride and cholesterol ester under different experimental conditions. The effect of storage temperature on the stability of the hydrolytic activity, and optimal conditions of temperature and time of maximal activity were determined. The optimal conditions for maximal lipolytic activity were found to be 40–50 °C and 1 h. The activity released to the medium by 1 mg cells for 1 h at 40 °C was stated as the enzyme released unit (ERU). The protein fraction of MW > 50 kDa obtained by ultrafiltration of the medium, was active on the three substrates assayed, and it showed a non-specific hydrolytic activity on both the 1- and 2-acyl esters either in the neutral glyceride or in the phosphoglyceride. A protein of M _r approx. 75 kDa was the only one that showed esterase activity. The crude medium, stored at –15 °C, maintained its initial hydrolytic activity on triacylglycerol for at least 42 days, though when it was kept for 10 days at 4 °C, the activity fell to 50%. Kinetic parameters using substrates such as triolein (TO), dipalmitoyl phosphatidylcholine (DPPC) and cholesteryl oleate (ChoO), were comparatively evaluated. The activity of the enzyme in the hydrolysis of TO showed the highest values, whereas the maximal specific activities were less when the enzyme was assayed against DPPC and ChoO.     

Enhanced protein recovery after electrotransfer using square wave alternating voltage

Protein identification is becoming a complement to the available fully sequenced genomes. To meet the challenge, newly developed techniques for high throughput protein identification using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and peptide mass fingerprint are needed. Two years ago, a parallel protein digestion process was proposed. It provided a collecting polyvinylidene difluoride (PVDF) membrane able to be scanned by MALDI. Acquired data were used to recreate a virtual multidimensional image. Voltage used during this protein electroblotting technique was an unusual square wave alternative voltage (SWAV). The goal of the current study is to evaluate quantitatively the efficiency of the SWAV compared with a classical electroblot process on intact proteins. The effect of the pulsed electric field and the buffer composition were compared to a standard continuous transblotting process defined as the gold standard. Combination of the pulsed asymmetric electric field with 3-(cyclohexylamino)-1-propane-sulfonique acid (CAPS) buffers showed an average 65% increase of protein recovery. Moreover, a strongest effect is observed for high M(r) proteins. In conclusion, the present study highlighted a positive influence of the "shaking" effect of the asymmetric alternative voltage on gel protein extraction.     

Egasyn, a protein which determines the subcellular distribution of beta-glucuronidase, has esterase activity

The glycoprotein egasyn complexes with and stabilizes precursor beta-glucuronidase in microsomes of several mouse organs. Several observations indicate egasyn is, in addition, an esterase. Liver homogenates of egasyn-positive strains have specific electrophoretically separable esterases which are absent in egasyn-negative mice. These esterases react with anti-egasyn serum. A specific esterase was likewise complexed with immunopurified microsomal beta-glucuronidase. The esterases were, like egasyn and microsomal beta-glucuronidase, concentrated in the microsomal subcellular fraction. Egasyn which is not bound to beta-glucuronidase, which represents 80-90% of total liver egasyn, is not complexed with other liver proteins. Egasyn, therefore, specifically stabilizes beta-glucuronidase in microsomes. The esterase activity is inhibited by bis-p-nitrophenyl phosphate indicating it is a carboxyl esterase. Several possible functions of egasyn-esterase activity are discussed.