Egg yolk lipoproteins as substrates for lipases

Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.     

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y) = -0.912 + 0.007X; R2 = 0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.     

A novel fluorogenic substrate for the measurement of endothelial lipase activity

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.     

A simple activity staining protocol for lipases and esterases

A simple activity staining protocol for rapid detection and differentiation of lipases and esterases was developed based on pH drop due to fatty acids released following lipolysis. Though the detection of lipolysis as a function of drop in pH is not new, the present method has been made more sensitive by the judicious selection of the initial pH of the chromogenic substrate, which has been set near the end point of the dye so that even a slight drop in pH results in immediate color change. In the present case, the dye phenol red was taken, which has the end point at pH 7.3–7.4 where the color is pink. A slight drop due to fatty acid release results in yellow coloration. The assay has high reproducibility and can detect as low as 0.5 p-NPP enzyme units within 15 min. In addition, this method can be used for various lipidic substrates such as oils and tributyrin, making it suitable for both lipases and esterases.     

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.     

A new method for determination of elastolytic activity using (14C) labeled elastin and its application to leukocytic elastase.

A new, highly sensitive and specific assay for elastolytic activity is described which employs insoluble elastin randomly labeled with [¹⁴C]. The substrate was prepared by labeling amino groups of the protein in vitro with [¹⁴C] methyl groups by reductive alkylation. The substrate was used to quantitate elastolytic activity from human leukocytes and to compare leukocytic elastase with pancreatic elastase. Purified human leukocytic elastase was approximately one-fourth as active as pancreatic elastase. Similar difference between leukocytic elastase and pancreatic elastase activities was found when the enzymes were tested against succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, but not when t-BOC-L-alanine-p-nitrophenyl ester was used.     

Lipid-lipid interactions as regulators of carboxylester lipase activity

The hydrolysis of 1,3-dioleoylglycerol and related substrates by mammalian pancreatic carboxylester lipases was studied. Mixed lipid films of substrates with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at the argon-buffer interface were exposed to relatively high levels of monomeric porcine pancreatic carboxylester lipase for a brief period. With either 1,3-dioleoylglycerol, 1,2-dioleoylglycerol, trioleoylglycerol, or oleoylmethanol as a substrate, the percentage of substrate hydrolysis increased abruptly from near zero to near 100% with increasing proportion of substrate in the film. The phospholipid was not hydrolyzed. Using 1,3-dioleoylglycerol as the substrate with either the dimeric, porcine pancreatic carboxylester lipase, human pancreatic carboxylester lipase, or human milk bile salt-stimulated lipase gave results identical to those obtained with the porcine monomer. Hydrolysis of 1,3-dioleoylglycerol by porcine monomeric carboxylester lipase was independent of the initial surface pressure of the film. However, a strong correlation was observed between hydrolysis and interfacial lipid composition at all surface pressures, even if bulk 1,3-dioleoylglycerol was also present. The ultrasensitive dependence of hydrolysis on interfacial lipid composition, i.e. lipid-lipid interactions, suggests that such "switching" may contribute to the regulation of diacylglycerol levels in cells where they function in signal transduction.     

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.     

Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.     

Identification of a UDP-Gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187

A novel acceptor substrate for galactosyltransferase was synthesized containing GlcNAcalpha-pyrophosphate, covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-Phenyl). The new substrate was used to develop an assay for a galactosyltransferase activity from Escherichia coli strain VW187 that is involved in lipopolysaccharide synthesis and has not been studied by others. We showed that Gal was transferred from UDP-Gal to the novel acceptor substrate. This was a significant improvement over our previous preliminary assays of the enzyme using endogenous substrate, and showed that these synthetic substrates are useful for assaying enzymes that utilize lipid-bound substrates in O-chain synthesis in Gram-negative bacteria.     

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     

Lipoprotein lipases and vitellogenins in relation to the known three-dimensional structure of pancreatic lipase

A 106-residue region of high similarity between lipoprotein/pancreatic/hepatic lipases and Drosophila vitellogenins encompasses four beta-strands with all residues but one strictly conserved or conservatively replaced between the structures, and enclosing the putative active site Ser-152. The properties suggest a common folding pattern but the region probably does not function as an 'interface recognition site' in the lipases, although it might well bind fatty acid esters of ecdysteroids or single lipid molecules in the vitellogenins. C-terminally of this 106-residue region, a surface loop ('flap') covers the active site. No residue within this loop is conserved through all lipases, but adjacent segments exhibit 60-70% residue identity. Hepatic and lipoprotein lipases probably hydrolyze both soluble and emulsified substrates at the same site. They lack residues corresponding to a second active site postulated in pancreatic lipase to account for hydrolysis of soluble substrates. In addition, due to structural differences the flap could prevent entry of soluble substrate molecules into the active site of pancreatic lipase.     

A rapid and specific assay for Nα-acetyltransferase activity

A rapid and specific assay for N^α-acetyltransferase activity is described. This assay makes use of acidic precipitation for removal of products resulting from acetylation of endogenous substrates from the enzyme preparation and of reverse-phase HPLC for isolation of the N-terminally acetylated synthetic substrate. The virtue of the assay lies in its fast, not too laborious, and reasonably accurate determination of activity during isolation and purification of the enzyme.     

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.     

Fluorimetric Assay of the Activity of Extracellular Lipases of Pseudomonas fluorescens and Serratia marcescens

A fluorimetric assay was carried out on the activity of extracellular lipase concentrations obtained from Serratia marcescens and Pseudomonas fluorescens using as substrates fatty acyl esters of 4-methylumbelliferone (4-methylumbelliferone elaidate, 4-methylumbelliferone nonanoate, 4-methylumbelliferone butyrate, 4-methylumbelliferone palmitate and 4-methylumbelliferone oleate) at pH 4.0, 6.0, 8.0 and 10.0. The Ser. marcescens and Ps. fluorescens were cultured in Pope and Skerman's basal medium (Skerman 1957) supplemented with 0.5% (w/v) of a commercial medium. The extracellular lipases were isolated and purified by ammonium sulphate precipitation. The assay was carried out by relating the fluorescent intensity emitted by two lipase concentrations on five substrates against four standard curves. These standard curves were prepared by estimating the intensity of fluorescence given by varying dilutions of 4-methylumbelliferone at the four pH levels. The results indicated that the oleic ester of 4-methylumbelliferone was a suitable substrate at pH 8.0 and pH 10.0. These pH values were also optimum for fluorescent intensity on substrates of 4-methylumbelliferone elaidate, 4-methylumbelliferone butyrate and 4-methylumbelliferone palmitate. However, on substrate 4-methylumbelliferone nonanoate, the optimum pH was 4.0.     

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.     

Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.     

A spectrophotometric assay for lipase activity utilizing immobilized triacylglycerols.

New substrates for the determination of lipase activity have been developed. Triacylglycerols were immobilized by adsorption on an appropriate carrier or adsorbent yielding a lipase substrate in a powder form. The adsorbed triacylglycerols were easily hydrolyzed by lipases present in a reaction mixture. The released fatty acids were extracted with benzene and converted to the corresponding Cu (II) salts (copper soaps) which were measured spectrophotometrically.     

Effects of glucose on lipase activity

To establish the utility of lipase as a biocatalyst, the effects of glucose on the hydrolysis activities of lipase were investigated. Among 13 kinds of lipase from microorganisms, 6 lipases were inhibited in hydrolysis up to 50% of the original activities by 10 mM glucose. The activities of other microbial lipases and 2 kind of porcine pancreatic lipases were not affected by the addition of glucose. Six lipases that were sensitive to glucose were modified by a synthetic detergent. After they were converted to modified lipases, they were not inhibited by glucose. Even at 20 mM glucose, each modified lipase retained more than 95% activity compared with that in the absence of glucose. In the modified lipase, the detergent attached to the lipase molecule would disturb the access of glucose to the enzyme. To detect the interaction between lipase and glucose, the fluorescence of tryptophan was traced. The fluorescence intensities of lipases that were inhibited by glucose depended on the concentration of glucose, suggesting that glucose induced some structural change in the lipase molecule.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.