If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.
In vitro enzymatic assays are a major source of information about the characteristics of enzymes. In particular the determination of functional parameters relies on data in well-defined in vitro systems. Enzymes of lipid metabolism have been investigated with radiolabeled substrates for decades (
). They represent ideal tracers that display all the characteristics of the unlabeled compound, but their synthesis or use can be laborious, requires special equipment, is often subject to special regulations, and the quantification of signal can be tedious. In recent times, fluorescently tagged lipids have been developed as an alternative to radioactive labeling. They are convenient and sensitive reporters, but in general contain spacious tags of high impact on the structure, likely to influence specificity and kinetic parameters of enzymatic reactions. Although there are reports of assays with unchanged affinity of the enzyme to fluorescently labeled substrates, notably nitrobenzoxadiazole (NBD)-sphingolipids [e.g., NBD-sphinganine (
)] the applicability of a substrate has to be validated thoroughly. For instance, when the boron dipyrromethene (BDP) analog, BDP-sphingosine-1-phosphate, was developed as a more photostable and hydrophobic alternative to NBD-sphingosine-1-phosphate, the BDP-, but not the NBD-derivate displayed a higher Km value than the natural substrate (
), and especially the copper-catalyzed Huisgen 1,3-dipolar cycloaddition between a terminal alkyne group and an azide, provide new possibilities for the bioorthogonal labeling of molecules like amino acids (
). In addition to these applications, it is especially worthwhile to test the feasibility of click-based in vitro assays in lipid metabolism, for which other labeled substrates often are not readily at hand.
As the alkyne moiety is a small, neither overly hydrophobic nor hydrophilic label, a good affinity of enzymes to alkyne-modified substrates can be expected. For the first time this study addresses the usage of alkyne lipids in enzymatic assays through the determination of Michaelis-Menten kinetics for these substrates. Here we describe a click-based in vitro enzymatic assay to study enzymes of lipid metabolism, notably from the families of lysophosphatidic acid acyltransferases (LPAATs), lysophosphatidylcholine acyltransferases (LPCATs), and ceramide synthases (CerSs).
MATERIALS AND METHODS
The chemical syntheses of the alkyne lipids are available in the supplementary material to this study, or were described previously (
Escherichia coli culture and preparation of microsomal fractions
E. coli (strain RosettaTM 2 pLysS, Merck Millipore) cultured in Luria Broth at 37°C to an OD600 of 1.6 were harvested by centrifugation at 3,000 g for 10 min. Microsomal fractions were prepared according to Lewin, Wang, and Coleman (
), frozen in liquid nitrogen and stored at −80°C. The protein concentration in the microsomes was determined with the BioRad Bradford assay kit using BSA as a standard.
Cell culture and preparation of cell lysate
HuH7 cells were grown in RPMI 1640 (PAN Biotech P04-17500) supplemented with 10 mM HEPES, 0.1 mM nonessential amino acids, 2 mM l-glutamine, 10% FBS, at 5% CO2. Cells were washed, scraped into ice-cold buffer (20 mM HEPES/NaOH, pH 7.0, 200 mM sucrose) and homogenized in a cooled EMBL cell cracker (HGM, Heidelberg, Germany) with five double strokes and a maximum clearance of 18 μm. The lysate was centrifuged at 500 g for 5 min at 4°C, the supernatant frozen in liquid nitrogen and stored at −80°C. The protein content of the lysate was determined with the bicinchoninic acid assay kit (Pierce) using BSA as a standard.
Animals and preparation of tissue microsomal fractions
Membrane fraction samples were prepared of the brain, liver, and kidneys of C57BL/6 wild-type (+/+) and CerS2-deficient mice (−/−) (one individual each, littermates, 13–14 weeks old) according to the protocol described by Imgrund et al. (
), frozen in liquid nitrogen and stored at −80°C. A total lysate of wild-type liver was also prepared. The protein content of the microsomal fractions, and of liver lysate was determined with the bicinchoninic assay kit (Pierce) using BSA as a standard.
All assays were performed in 1.5 ml reaction tubes in a total volume of 100 μl. The reaction was started by addition of the prewarmed reaction mix to the enzyme preparation and the tubes were incubated in a heating block (Eppendorf Thermomixer comfort) under shaking (1,100 rpm). Incubation times and the amount of enzyme preparation were optimized for each assay (data not shown) to ensure substrate saturation and linearity of the reaction rate with regard to time and enzyme amount.
For the kinetic studies, different concentrations of 1-(nonadec-9-cis-en-18-ynoyl)-sn-glycero-3-phosphate (alkyne-OLPA) and 50 μM oleoyl-CoA, or various concentrations of 1-palmitoyl-lysophosphatidic acid (PLPA) and 50 μM nonadec-9-cis-en-18-ynoyl CoA (alkyne-oleoyl-CoA) were incubated with 2.06 μg (protein) E. coli microsomes in LPAAT buffer (
) [60 mM Tris/HCl, pH 7.5, 3 mM MgCl2, 0.6 mg/ml lipid-free BSA (Applichem A0848)] for 5 min at 30°C. For the measurement of the acyl-CoA specificity (Fig. 2D, 5 min incubation) and the TLC displayed in the results section (Fig. 2C, 10 min incubation), 4 μM alkyne-OLPA and 100 μM acyl-CoA were used.
For the kinetic studies, different concentrations of 1-(nonadec-9-cis-en-18-ynoyl)-sn-glycero-3-phosphocholine (alkyne-OLPC) or palmitoyl-lyso-propargyl-phosphatidylcholine (PLpPC) and 150 μM oleoyl-CoA were incubated with 5 μg (protein) HuH7 lysate for 15 min at 30°C in LPCAT buffer (60 mM Tris/HCl, pH 7.4, 3 mM MgCl2, 0.6 mg/ml lipid-free BSA). For the TLC that shows the setup of the assay (Fig. 3B), 20 μM lysophosphatidylcholine, 50 μM oleoyl-CoA, and 5 μg (protein) HuH7 lysate were incubated for 30 min.
The kinetic measurements were performed with different concentrations of (2S,3R)-2-aminooctadec-17-yn-1,3-diol (alkyne-sphinganine), 50 μM nervonoyl(24:1)-CoA, and 5 μg (protein) mouse wild-type liver microsomes. The assay was incubated for 20 min at 37°C in CerS buffer (20 mM HEPES/KOH, pH 7.4, 25 mM KCl, 250 mM sucrose, 2 mM MgCl2, 0.34 mg/ml lipid-free BSA) (
). For the TLC that shows the setup of the assay (Fig. 4B), 20 μM alkyne-sphinganine, 50 μM nervonoyl- or stearoyl-CoA, and 20 μg (protein) liver wild-type microsomes were used. For screening various tissues for CerS activity (Fig. 4D), 10 μg (protein) brain, kidney, or liver microsomes of wild-type or CerS2−/− mice were incubated with 20 μM alkyne-sphinganine and 50 μM acyl-CoA.
Incubation of additional substrates with mouse liver microsomes or lysate
Incubation of 1-(nonadec-9-cis-en-18-ynoyl)-monoacylglycerol (alkyne-1-OMAG) (50 μM) was performed in LPCAT buffer for 30 min at 30°C with oleoyl-CoA or palmitoyl-CoA (100 μM) and mouse liver microsomes (50 μg protein). Incubation of 1-(16-heptadecanoyl)-2-((5Z,8Z,11Z,14Z)-5,8,11,14-eicosatetraenoyl)-sn-glycero-3-phosphate (alkyne-PAPA) (50 μM) was performed with different amounts of mouse total liver lysate (0, 2, 10, and 50 μg protein) in ammonium acetate buffer (60 mM ammonium acetate, pH 7.0, 0.6 mg/ml lipid-free BSA) for 30 min at 30°C. Alkyne-palmitoylethanolamide or alkyne-oleoylethanolamide (50 μM) were incubated in LPCAT buffer at 30°C with or without the addition of mouse liver microsomes (10 μg protein) for different timespans (2–30 min).
Lipid extraction, click reaction, and TLC
All assay reactions were stopped by the addition of 500 μl chloroform/methanol 1/3 (v/v) to the assay mixture. Samples were then incubated for 5 min in an ultrasonic bath and 500 μl 1% acetic acid were added, followed by vortexing and centrifugation at 14,000 g for 2 min. One hundred microliters of the chloroform phase were transferred to a new 1.5 ml reaction vessel. The aqueous phase was again extracted with 200 μl chloroform. The combined organic phases were dried in a speed-vac and the click reaction performed as described previously (
): To the dried extracts, 7 μl chloroform were added to redissolve lipids. Then 30 μl click reaction mix (10 μl of 2 mg/ml 3-azido-7-hydroxycoumarin, 250 ul of 10 mM [acetonitrile]4CuBF4 in acetonitrile, 850 μl ethanol) were added and the tubes incubated in a heating block (Eppendorf Thermomixer comfort) at 43°C without shaking until all solvent was condensed under the lid (3 h).
After brief centrifugation, addition of 30 μl chloroform, and 5 min incubation in an ultrasonic bath, the samples were applied onto TLC silica plates (Merck 1.05721.0001). Plates were developed in the respective solvent system: a) for all assays except CerS: first chloroform/methanol/water/acetic acid 65/25/4/1 for 13 cm, then hexane/ethyl acetate 1/1 for 18 cm, with gentle drying between the two solvents; or b) for the CerS assay: chloroform/methanol/water 80/10/1 (
Diluted solutions (0.5 μM) of 1,2-di-(nonadec-9-cis-en-18-ynoyl)-sn-glycero-3-phosphate (alkyne-OOPA), alkyne-18:0-dihydroceramide (dhCer), alkyne-PC, and propargyl-phosphatidylcholine (pPC) in ethanol were prepared freshly, 10, 20, or 40 μl of this solution added to a reaction vessel, the solvent evaporated, and the lipids dissolved in the appropriate assay buffer (see above). After the addition of heat-inactivated enzyme preparation, the reaction mix was incubated according to the respective assay protocol and subjected to extraction, click reaction, and TLC. Quantification against alkyne-oleate was performed as given below, using the same molar amounts of alkyne-oleate as for the extracted lipids.
Detection and quantification
Shortly before fluorescence detection, the dry TLC was soaked for 5 s in 4% (v/v) N,N-diisopropylethylamine in hexane and excess solvent was allowed to evaporate in a hood. The system used for the imaging of the TLC plates and image quantification was described earlier (
). Briefly, standard LEDs (10 × 1 W 420 nm LEDs; Roithner Lasertechnik, Vienna, Austria) filtered through a colored glass filter (HEBO V01, Hebo Spezialglas) were used for excitation. Images were acquired with a Rolera MGI plus electron multiplying charge-coupled device (EMCCD) camera (Decon Science Tec), equipped with a 494/20 (channel for detection of the coumarin fluorescence) and 572/28 (channel for detection and correction of background fluorescence) bandpass emission filter wheel, all under control of GelPro analyzer software (Media Cybernetics). Note that the high sensitivity of the EMCCD camera (which is part of our electrochemiluminescence Western blot detection system) is not necessary to record the images, which typically can be seen already by visual inspection, but its large dynamic range is advantageous for image quantification. We also use a system with a much simpler camera with good results. Fluorescent signals were correlated to the lipid amount detected by drying two different defined amounts of alkyne-oleate solution in the speed-vac, subjecting them to the click reaction, and applying them to separate lanes in the TLC. All signals of a TLC were quantified against the weighted mean signal of the alkyne-oleate signals.
Statistical analysis and nonlinear regression
Unless stated otherwise (extraction efficiency measurements), all data are presented as mean values ± standard deviations (n = 3). Michaelis-Menten kinetics were assessed in three independent measurements, a representative graph is shown. For each measurement, the kinetic constants Km and Vmax were calculated from the data using nonlinear regression in Microsoft Excel (
), and the mean value and standard deviation of these three calculations are given.
RESULTS AND DISCUSSION
Assay setup and method of quantification
The general workflow, as outlined in Fig. 1, consists of the enzymatic reaction using alkyne substrate, lipid extraction, and reaction of the alkyne moieties with the fluorogenic dye 3-azido-7-hydroxycoumarin in a quantitative copper(I)-catalyzed cycloaddition. After separation by TLC, lipids are analyzed by fluorescence detection.
The high sensitivity and wide linear dynamic range of this procedure regarding the click reaction, TLC, and imaging were established earlier (
) and are comparable to methods using [3H]-labeled lipids. For an accurate quantification of the assay products, a reliable and near complete recovery is necessary. To determine the efficiency of the lipid extraction protocol, defined amounts of alkyne lipids that represent products of the LPAAT, LPCAT, or CerS assay were subjected to the standard procedures (in vitro enzymatic reaction, extraction, click reaction, detection), but using heat-inactivated enzyme preparations. The extraction procedure was evaluated by quantification of fluorescent signal against that of defined amounts of alkyne-oleate that had not been subjected to assay incubation and extraction. High recovery rates were measured for all tested lipids, alkyne-OOPA (97%), alkyne-dhCer (85%), alkyne-PC (88%), and pPC (82%), with good to very good reproducibility. This demonstrates the applicability of the extraction protocol (which had been optimized for polar lipids) for the assays performed in this study, and of the quantification method. Thus, an exact correlation of fluorescent signal to the amount of lipid produced in the assay is achieved.
LPAAT assay with alkyne-OLPA or alkyne-oleoyl-CoA
In E. coli, the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA) is catalyzed by the gene product of plsC, 1-acyl-sn-glycero-3-phosphate acyltransferase (
). To the best of our knowledge, kinetic constants have not been determined for this enzyme.
We established a LPAAT assay with alkyne-OLPA (Fig. 2A), the labeled analog of sn-1-oleoyl-lysophosphatidic acid using E. coli microsomes (Fig. 2B), and determined the constants Vmax and Km for this substrate (Fig. 2C, Table 1). Conversely, by applying alkyne-oleoyl-CoA as an acyl chain donor, we measured Vmax and Km for PLPA. The apparent Km for alkyne-OLPA (0.53 ± 0.18 μM) was significantly lower than for PLPA (3.64 ± 0.24 μM). Also, Vmax was apparently increased for alkyne-OLPA compared with PLPA, but the differences did not reach statistical significance due to large variations between different enzyme preparations. In other organisms, Km had previously been determined for LPAAT enzymes using radiolabeled substrates. A similar acyl chain specificity in the acceptor was found in Spinacia oleracea (
); with oleoyl-CoA as the donor, a Km of 5.3 μM was measured for [14C]1-oleoyl-lysophosphatidic acid (OLPA), and of 3.0 μM for [14C]PLPA. For the human LPAAT-α, a Km of 6.49 μM toward OLPA with [14C]palmitoyl-CoA as the acyl chain donor (
). Compared with palmitate and stearate, oleate is preferred by the E. coli enzyme also as the donor acyl-CoA (Fig. 2D) for the incorporation at the sn-2 position, in accordance with previous findings (
Our results demonstrate that alkyne-OLPA and alkyne-oleoyl-CoA are good substrates for the E. coli LPAAT. They indicate that the bacterial enzyme may have a lower Km for lysophosphatidic acids (especially OLPA) than the plant or mammalian enzymes investigated so far.
The two alkyne substrates described above represent the two strategies that are available for an acyltransferase assay with alkyne lipids, i.e., labeling of the acyl chain acceptor or the acyl chain donor. While the latter is the more general approach, enabling the investigation of all acyltransferase reactions with a relatively small set of labeled alkyne-acyl-CoAs, it suffers from the limited storage stability of acyl-CoAs and the frequent observation of significant amounts of labeled side products arising from both enzymatic and nonenzymatic conversion of the acyl-CoAs. In our experiments, the use of alkyne-oleoyl-CoA led to a few fluorescent signals derived from side reactions of the reactive compound including the hydrolysis to oleate. The labeled acceptors on the other hand expressed high specificity for the enzymatic reactions of interest. They essentially gave one or two strong product signals besides the fluorescent educt (Figs. 2B, 3B, 4B), which might be beneficial for automated image analysis. In addition, alkyne-labeled acyl acceptors show better stability for long-term storage.
LPCAT assay with alkyne-OLPC or PLpPC
Lysophosphatidylcholine (LPC) is converted to phosphatidylcholine (PC) by lysophosphatidylcholine acyltransferases. In mammals, four isoforms (LPCAT1–4) are known. HuH7 cells express LPCAT1 and LPCAT3, but not LPCAT2 (
). We synthesized two alkyne-labeled LPC species, i.e., side chain-labeled alkyne-OLPC and headgroup-labeled PLpPC (Fig. 3A). Note that because PLpPC is derived by enzymatic headgroup exchange from egg yolk PC, it contains a mixture of fatty acids at sn-1, dominated by palmitate (
). Figure 3B shows the application of the two substrates in a LPCAT assay on HuH7 lysate using oleoyl-CoA as the acyl chain donor. The Km value we measured (Fig. 3C, Table 1) for alkyne-OLPC (2.46 ± 1.62 μM) was very similar to that determined for palmitoyl-LPC with [14C]palmitoyl-CoA for murine recombinant LPCAT1 (2.3 μM) (
). PLpPC had a higher apparent Km (5.46 ± 2.23 μM), which might reflect a somewhat stronger effect of the alkyne label when incorporated in the headgroup of a phospholipid rather than at the terminus of an acyl chain. However, the differences between the Km values for alkyne-OLPC and PLpPC were not statistically significant and the differential acyl chains at the sn-1 positions have to be taken into account. In addition, we observed a significant difference (P < 0.05) in activity (Vmax) for the two substrates, 0.51 ± 0.08 nmol/(mg·min) for alkyne-OLPC compared with 0.30 ± 0.07 nmol/(mg·min) for PLpPC. On the basis of these results, alkyne-OLPC may be considered as the substrate of first choice for enzymatic assays compared with PLpPC. Nevertheless, the latter also displayed near to natural kinetic constants and allows for the direct combination of enzyme kinetics with microscopic imaging after labeling with propargyl-choline (
). We synthesized the new substrate alkyne-sphinganine (Fig. 4A) and employed it in a CerS assay on mouse liver microsomes using nervonoyl-CoA (Fig. 4B). In kinetic measurements in the same system, we determined an apparent Km of 2.29 ± 2.08 μM and a Vmax of 0.71 ± 0.38 nmol/(mg·min) for alkyne-sphinganine (Fig. 4C, Table 1). In the liver, CerS2 is the dominant isoform with activity toward long-chain acyl-CoAs; hence the activity we measured in this assay may almost solely be attributed to CerS2 (
) reported Km values in the range of 2–5 μM for all six (murine or human) CerS isoforms, even for those which use multiple acyl-CoAs. That study reported a Km of 4.8 ± 0.4 μM for human Cers2 with lignoceroyl (24:0)-CoA. Kim et al. (
) developed a fluorescent assay with NBD-sphinganine and nervonoyl-CoA and found Km to be comparable to natural sphinganine (3.61 ± 1.86 μM vs. 3.05 ± 0.81 μM, respectively). However, Vmax was elevated for the NBD derivative.
CerS2 is expressed in various tissues besides the liver, with high mRNA levels in the kidney and moderate levels in the brain (
) created a CerS2-deficient mouse and measured the CerS activity toward tritiated sphinganine and various acyl-CoAs (16:0, 18:0, 20:0, 22:0, and 24:1) in brain and liver microsomes. To demonstrate that alkyne-sphinganine can be used as a convenient alternative substrate for such applications, we performed a similar small screen with brain, liver, and kidney microsomes and several acyl-CoAs (16:0, 18:0, 18:1, and 24:1). Figure 4D shows fluorescent images of the TLC plates of this pilot study. Like Imgrund et al. (
), we observed a strong activity toward 24:1-CoA in the wild-type liver and a weak one in the brain. We also measured a strong 24:1 activity in kidney microsomes. For all tissues, 24:1-CoA was not converted to dhCer in the CerS2-deficient samples. For the acyl-CoAs of shorter chain length, the CerS activity was similar for wild-type and −/− mice, reflecting that it was not derived from CerS2, but other isoforms. We detected activity toward 16:0-CoA in kidney (moderate) and liver (weak), but not in brain. Weak activity was also found for 18:0-CoA in brain and liver, both in wild-type and −/− mice. 18:1-CoA was converted to dhCer in kidney and very little in liver. Because we show here that alkyne-sphinganine is a useful substrate to screen for CerS activity in tissue samples, more detailed quantitative investigations can be carried out in the future.
Application of the method in assays for other lipid-modifying enzymes
We utilized mouse liver microsomes or mouse liver lysate to demonstrate the principal feasibility of assays with four additional examples of alkyne-labeled substrates (Fig. 5). All substrates were converted by one or more enzymes in the liver fractions to alkyne-labeled products.
Deacylation of alkyne-1-OMAG (structure depicted in Fig. 5A) by monoacylglycerol lipases (MAGLs) yielded alkyne-oleate (Fig. 5A, lane 1). Upon addition of oleoyl-CoA or palmitoyl-CoA, alkyne-1-OMAG was acylated at the sn-2 position to form diacylglycerol (DAG) (Fig. 5A, lane 2+3) by monoacylglycerol acyltransferase (MGAT) activities, presumably the murine MGAT1 and lysophosphatidylglycerol acyltransferase 1 (
). In parallel, we observed the release of alkyne-palmitate from the sn-1 position, either by a phospholipase 1 activity acting on the labeled PA, or by a DAG lipase activity acting on the released DAG.
N-oleoylethanolamide and N-palmitoylethanolamide are bioactive lipids that are cleaved by the enzymes fatty acid amide hydrolase (FAAH) (
). We were able to survey the hydrolysis of the fatty acid amides alkyne-oleoylethanolamide and alkyne-palmitoylethanolamide in a time-course experiment (Fig. 5C) upon incubation with mouse liver microsomes. Several enzymatic assays are reported to follow this reaction [see (
)]. We will conduct further studies concerning the kinetic properties of these alkyne-labeled substrates, which would expand the existing techniques by a very convenient and direct assay procedure.
The various substrates discussed above therefore demonstrate how the scope of the assay can be extended to more acyltransferases (MGAT), lipases (MAGL, phospholipases A1 and A2) and other hydrolases (PA phosphatase, FAAH). This general applicability will prove useful for the kinetic characterization of many enzymes in lipid metabolism.
Scope and limitations of the method
All alkyne lipids that we tested in this pilot study were used as substrates by lipid-modifying enzymes. No substantial shift in affinity (Km) of the enzymes compared with the natural substrates was detected in our kinetic studies. We thus introduce enzymatic assays with alkyne lipids as a reliable and convenient alternative to fluorescent or radioactive assays.
As with the latter two methods, the TLC separation in our assay does not achieve species resolution of the lipids. This can however be overcome by a subsequent mass-spectrometric analysis [compare (
), as does the headgroup cleavage of pPC to PA by phospholipase D.
Our studies suggest that the alkyne label does not generally interfere with the affinity of enzymes to the substrates, especially if the alkyne label is attached to the terminus of the alkyl chain. Such fatty acids of different length and unsaturation are synthetically available (
) and can serve as a basis of a versatile toolbox of tailor-made lipid substrates with the acyl chain(s) of choice at defined sn-positions, including phospholipids, lysophospholipids, acyl-CoAs, and acylglycerols, using standard chemical and enzymatic procedures. Similar to this strategy, alkyne-sphingolipids can be synthesized from alkyne-sphinganine. Hence, we expect alkyne lipids to become more and more commercially available in the coming years, both as substrates for enzymatic assays as well as standards for identification after TLC separation. The migration behavior of lipids coupled to the coumarin dye is shifted, but, in general, the separation is possible with the same solvent mixes as for radiolabeled lipids or slightly adapted versions thereof and the retention factors of the substances usually are in the regular order. The adaptation of radiolabeled assays to the use of alkyne lipid substrates should therefore be neither costly nor overly time-consuming.
Alkyne lipids are promising substrates for use in in vitro enzymatic assays, as they are inexpensive, versatile, convenient and, as we show here for the first time, display the kinetic characteristics of the natural substrates.
The authors would like to thank Christiane Kremser and Prof. Dr. K. Willecke for providing the mice used in the CerS assay.