Probes for Lipid Metabolism and Signaling—Section 17.4

Lipids and lipid metabolites are abundant in cells and have both a structural function and a role in cell regulation. Phospholipases, in particular, play an important part in cellular signaling processes via the generation of second messengers such as diacylglycerols, arachidonate and inositol 1,4,5-triphosphate (Ins 1,4,5-P₃, I3716; Calcium Regulation—Section 17.2). In addition, phospholipase A₂ activation is a key step in inflammation processes, and phospholipase A₂ plays a major role in the pathogenesis of acute respiratory distress syndrome (ARDS), making this class of enzymes important therapeutic targets. Enzymatic processing of sphingolipids and phospholipids produces a wide range of physiological and pathological effects, including apoptosis, inflammatory responses, cell differentiation and proliferation.

Phospholipases are classified according to the cleavage site on the phospholipid substrate (Figure 17.23). There are at least three types of fluorescence-based phospholipase detection methods:

• Continuous methods, which permit direct fluorometric monitoring of enzymatic activity using self-quenching or excimer-forming probes
• Methods that continuously detect nonfluorescent product formation from natural phospholipids, such as detection of the fatty acid component with our ADIFAB reagent or measurement of choline with our Amplex Red Phospholipase Assay Kits
• Discontinuous methods, which require resolution of fluorescent substrates and products by TLC, HPLC or other separation techniques

Fluorescence-based phospholipase assays—Table 17.2 summarizes Molecular Probes' products for fluorescence-based phospholipase assays. Other applications for our wide range of fluorescent phospholipids are described in Probes for Lipids and Membranes—Chapter 13.

Figure 17.23 Cleavage specificities of phospholipases. R1 and R2 are typically saturated or unsaturated aliphatic groups. The polar head group R3 can be choline, ethanolamine, glycerol, inositol, inositol phosphate, serine or other alcohols.

The importance of phospholipases in cellular signaling, lipid metabolism, inflammatory responses and pathological disorders related to these processes has stimulated demand for fluorescence-based enzyme activity monitoring methods. Several of the fluorogenic phospholipase A substrates described here are designed to provide continuous monitoring of phospholipase A activity in purified enzyme preparations, cell lysates and live cells; applications of some of these substrates extend as far as in vivo small animal imaging. The phospholipase A substrates are generally dye-labeled phospholipids of two types—glycerophosphocholines with BODIPY dye–labeled sn-1 and sn-2 acyl or alkyl chains and glycerophosphoethanoloamines with BODIPY dye–labeled acyl chains and dinitrophenyl quencher–modified head groups. These structural variations determine specificity for phospholipase A₁ (which hydrolyzes the sn-1 ester linkage between phospholipids and fatty acids) versus phospholipase A₂ (which hydrolyzes the sn-1 ester linkage between phospholipids and fatty acids), and the fluorescence response associated with enzymatic cleavage of the substrate (Fluorescence-based phospholipase assays—Table 17.2).

PED-A1 Phospholipase A₁ Substrate

PED-A1(A10070, ) is a fluorogenic substrate designed to provide specific, real-time monitoring of phospholipase A₁ activity in purified enzyme preparations, cell lysates and live cells. PED-A1 is comprised of a dinitrophenyl quencher–modified glycerophosphoethanolamine head group and a green-fluorescent BODIPY FL dye–labeled acyl chain at the sn-1 position. Upon cleavage by phospholipase A₁, PED-A1 exhibits an increase in green fluorescence (measured at exitation/emission = 488/530 nm) as a result of the uncoupling of the quenched state. Phospholipase A₁ specificity is imparted by the placement of the BODIPY FL acyl chain in the sn-1 position and by incorporation of an acyl group with an enzymatic-resistant (noncleavable) ether linkage in the sn-2 position.

EnzChek Phospholipase A₁ Assay Kit

The EnzChek Phospholipase A₁ Assay Kit (E10219, E10221) provides a simple, fluorometric method for continuous monitoring of phospholipase A₁ activity based on the phospholipase A₁–specific PED-A1 substrate (A10070, described above). The EnzChek Phospholipase A₁ Assay Kit can detect phospholipase A₁ activity at 0.04 U/mL or lower (Figure 17.44). This microplate-based assay is well suited for rapid and direct analysis of phospholipase A₁ in purified enzyme preparations and cell lysates using automated instrumentation, as well as for characterizing phospholipase A₁ inhibitors.

Each EnzChek Phospholipase A₁ Assay Kit (2-plate size, E10219; 10-plate size, E10221) provides:

• PED-A1 phospholipase A₁ substrate
• Phospholipase A₁ (Lecitase Ultra)
• Concentrated phospholipase A₁ reaction buffer
• Dioleoylphosphatidylcholine (DOPC)
• Dioleoylphosphatidylglycerol (DOPG)
• Dimethylsulfoxide (DMSO)
• Detailed assay protocols (EnzChek Phospholipase A1 Assay Kit)

The 2-plate assay kit provides sufficient reagents for 200 reactions in 96-well microplates at a volume of 100 µL per well or 800 reactions using low-volume 384-well microplates at a volume of ≤25 µL per well. The 10-plate assay kit provides sufficient reagents for 1000 reactions in 96-well microplates at a volume of 100 µL per well or 4000 reactions using low-volume 384-well microplates at a volume of ≤25 µL per well.

Figure 17.44 Detection of phospholipase A1 (PLA1) using the EnzChek Phospholipase A1 Assay Kit (E10219, E10221). PLA1 reactions were run at ambient temperature with liposomes for 30 minutes according to the assay protocol provided, and fluorescence emission was measured using 460 nm excitation on a Spectra Max M5 (Molecular Devices). Background fluorescence determined for the no-enzyme control reaction has been subtracted.

Red/Green BODIPY PC-A2 Ratiometric Phospholipase A₂ Substrate

Red/Green BODIPY PC-A2 (A10072, ) is a ratiometric fluorogenic substrate designed to provide selective, real-time monitoring of phospholipase A₂ activity in purified enzyme preparations, cell lysates and live cells. Cleavage of the BODIPY FL pentanoic acid substituent at the sn-2 position results in decreased quenching by fluorescence resonance energy transfer (FRET) of the BODIPY 558/568 dye attached at the sn-1 position. Thus, upon cleavage by phospholipase A₂, Red/Green BODIPY PC-A2 exhibits an increase in BODIPY FL fluorescence, detected from 515–545 nm (Figure 17.45). The FRET-sensitized BODIPY 558/568 fluorescence signal is expected to show a reciprocal decrease; in practice, however, this longer-wavelength fluorescence may show a decrease or a slight increase, depending on the formulation of the substrate and the instrument wavelength settings. The ratiometric detection mode of this substrate (emission intensity ratio at 515⁄575 nm with excitation at ~460 nm) allows measurements of phospholipase A₂ activity that are essentially independent of instrumentation and assay conditions. The dual-emission properties of this substrate also provide the capacity to localize the lysophospholipid and fatty acid products of the phospholipase A₂ cleavage via their distinct spectroscopic signatures in imaging experiments. This product offers an alternative to bis-BODIPY FL C₁₁-PC (B7701, see below) that both is selective for phospholipase A₂ and allows ratiometric measurements.

Figure 17.45 Fluorescence emission spectra (excitation at 480 nm) of Red/Green BODIPY PC-A2 phospholipase A2 substrate (A10072) incorporated in liposomes with addition of bee venom phospholipase A2 at ambient temperature.

EnzChek Phospholipase A₂ Assay Kit

The EnzChek Phospholipase A₂ Assay Kit (E10217, E10218) provides a simple, fluorometric method for continuous monitoring of phospholipase A₂ activity based on the phospholipase A₂–selective Red/Green BODIPY PC-A2 (A10072, described above). This phospholipase A₂ assay can be used in an intensity-based detection mode, by following the fluorescence increase at ~515 nm, or in a ratiometric-based detection mode, by following the changes in the emission intensity ratio at 515/575 nm with excitation at ~460 nm (Figure 17.46). The EnzChek Phospholipase A₂ Assay Kit can detect bee venom phospholipase A₂ activity at 0.05 U/mL or lower (Figure 17.46). This microplate-based assay is well suited for rapid and direct analysis of phospholipase A₂ in purified enzyme preparations and cell lysates using automated instrumentation, as well as for characterizing phospholipase A₂ inhibitors.

Each EnzChek Phospholipase A₂ Assay Kit (2-plate size, E10217; 10-plate size, E10218) provides:

• Red/Green BODIPY PC-A2 phospholipase A₂ substrate
• Phospholipase A₂ from honey bee venom
• Concentrated phospholipase A₂ reaction buffer
• Dioleoylphosphatidylcholine (DOPC)
• Dioleoylphosphatidylglycerol (DOPG)
• Dimethylsulfoxide (DMSO)
• Detailed assay protocols (EnzChek Phospholipase A2 Assay Kit)

The 2-plate assay kit provides sufficient reagents for 200 reactions in 96-well microplates at a volume of 100 µL per well or 800 reactions using low-volume 384-well microplates at a volume of ≤25 µL per well. The 10-plate assay kit provides sufficient reagents for 1000 reactions in 96-well microplates at a volume of 100 µL per well or 4000 reactions using low-volume 384-well microplates at a volume of ≤25 µL per well.

Figure 17.46 Detection of phospholipase A2 (PLA2) using the EnzChek Phospholipase A2 Assay Kit (E10217, E10218). PLA2 reactions were run at ambient temperature with liposomes for 10 minutes according to the assay protocol provided, and fluorescence emission was measured using 460 nm excitation on a Spectra Max M5 (Molecular Devices). Background fluorescence determined for the no-enzyme control reaction has been subtracted. Top panel shows ratiometric-based (515/575 nm) detection mode; bottom panel shows intensity-based (515 nm channel) detection mode. Background fluorescence determined for the no-enzyme control reaction has been subtracted for each value.

PED6 Phospholipase A₂ Substrate

PED6 (D23739, ) is a fluorogenic substrate for phospholipase A₂ incorporating a BODIPY FL dye–labeled sn-2 acyl chain and a dinitrophenyl quencher–labeled head group (Figure 17.24). Cleavage of the dye-labeled acyl chain by phospholipase A₂ eliminates the intramolecular quenching effect of the dinitrophenyl group, resulting in a corresponding fluorescence increase. Continuous kinetic assays show PED6 to be a good substrate for both secreted and cytosolic phospholipase A₂ and platelet-activating factor acetylhydrolase. PED6 has been used by Steven Farber and co-workers for in vivo analysis of intestinal lipid metabolism in zebrafish larvae as a basis for identifying and screening mutant phenotypes (). PED6 is also useful for high-throughput screening of potential phospholipase A₂ inhibitors or activators.

Other BODIPY Dye Phospholipase A Substrates

The bis-BODIPY phospholipase A substrate—bis-BODIPY FL glycerophosphocholine (bis-BODIPY FL C₁₁-PC, B7701)—has been specifically designed to allow continuous monitoring of phospholipase A action and to be spectrally compatible with argon-ion laser excitation sources. When this probe is incorporated into cell membranes, the proximity of the BODIPY FL fluorophores on adjacent phospholipid acyl chains causes fluorescence self-quenching (Figure 17.24). Separation of the fluorophores upon hydrolytic cleavage of one of the acyl chains by either phospholipase A₁ or A₂ results in increased fluorescence. Bis-BODIPY FL C₁₁-PC has been developed in collaboration with Elizabeth Simons, who has successfully employed it for flow cytometric detection of phospholipase A activity in neutrophils. Using bis-BODIPY FL C₁₁-PC and indo-1 simultaneously, researchers in the Simons lab at Boston University have demonstrated that a rise in intracellular Ca²⁺ precedes phospholipase A activation in immune complex–stimulated cells. More recently, bis-BODIPY FL C₁₁-PC has been used to detect phospholipase A₂ activation induced by tumor necrosis factor (TNF) and for imaging G-protein–coupled phospholipase A activity associated with plant cell plasma membranes.

Specificity for phospholipase A₂ versus phospholipase A₁ can be obtained using phospholipids with nonhydrolyzable, ether-linked alkyl chains in the sn-1 position. A 1-O-alkyl–substituted phospholipid containing the BODIPY FL fluorophore (D3771, ) is a useful substrate for a phospholipase A₂–specific chromatographic assay.

The singly labeled BODIPY phospholipase A₂ substrate—β-BODIPY FL C₅-HPC (D3803)—has been used to quantitatively delineate a discontinuous increase of Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) activity during zebrafish embryogenesis. The analytical method developed for this study uses a fluorescence image scanner to quantitatively detect the free BODIPY FL dye–labeled fatty acid generated by the action of cPLA₂ ().

Bis-Pyrenyl Phospholipase A Substrates

Our bis-pyrenyl phospholipase A probes (B3781, B3782) both emit at ~470 nm, indicating that their adjacent pyrene fluorophores () form excited-state dimers (Figure 13.8). Phospholipase A–mediated hydrolysis separates the fluorophores, which then emit as monomers at ~380 nm. These substrates have proven to be effective phospholipase A₂ substrates in model membrane systems (Fluorescence-based phospholipase assays—Table 17.2); however, it has been reported that 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (B3781) is highly resistant to degradation by phospholipases in human skin fibroblasts. 1,2-Bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine has been used in a sensitive, continuous assay for lecithin:cholesterol acyltransferase (LCAT). These probes have several other reported applications, including investigations of protein kinase C (PKC) interactions with lipids, DNA binding to liposomes and lipid dynamics.

Figure 13.8 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.

Singly Labeled Pyrenyl and NBD Phospholipase A₂ Substrates

Phospholipase A₂ activity has also been measured using phospholipids labeled with a single pyrene (H361, ; H3809, ; H3810) or NBD (N3786; N3787, ) fluorophore (Fluorescence-based phospholipase assays—Table 17.2). Because only the sn-2 phospholipid acyl chain is labeled, these probes can discriminate between phospholipase A₂ and phospholipase A₁ activity. To obtain a direct fluorescence response to enzymatic cleavage, sufficient phospholipid must be loaded into membranes to cause either intermolecular self-quenching (NBD-acyl phospholipids) or excimer formation (pyreneacyl phospholipids).

Pyrene-labeled acidic phospholipids—particularly the phosphoglycerol and phosphomethanol derivatives (H3809, H3810)—are preferred as substrates by pancreatic and intestinal phospholipase A₂, whereas labeled phosphocholine (H361, ) is preferred by phospholipase A₂ from snake venom. A very sensitive continuous assay that uses pyrene-labeled phosphomethanol (H3810) to measure mammalian cytosolic phospholipase A₂ and human nonpancreatic phospholipase A₂ has been described.

1-Octacosanyl-2-(1-pyrenehexanoyl)-sn-glycero-3-phosphomethanol (C₂₈-O-PHPM, O7703; ) was developed for monitoring phospholipase A₂ in serum because the bis-pyrenyl probes are not specific for phospholipase A₂ and can also produce false indications of activity due to interactions with serum proteins. The cleavage product of C₂₈-O-PHPM hydrolysis by phospholipase A₂—1-pyrenehexanoic acid (P3840, Fatty Acid Analogs and Phospholipids—Section 13.2)—cannot be directly distinguished from the substrate based on its fluorescence; however, this product can be readily resolved by liquid/liquid partition of the dye into aqueous methanol from chloroform/heptane.

ADIFAB and DAUDA: A Different View of Phospholipase A Activity

The ADIFAB fatty acid indicator (A3880, Figure 17.42) functions as a fluorescent sensor for the free fatty acid cleavage products of phospholipases. It does not require membrane loading and can be used to monitor hydrolysis of natural (rather than synthetic) substrates. Assaying lysophospholipase activity with ADIFAB yields sensitivity comparable to radioisotopic methods. Richieri and Kleinfeld have described a methodology for using the ADIFAB reagent to measure the activity of phospholipase A₂ on cell and lipid-vesicle membranes; their assay is capable of detecting hydrolysis rates as low as 10^–12 mole/minute. Displacement of dansylundecanoic acid (DAUDA, D94) from the rat intestinal fatty acid–binding protein I-FABP is the basis of another phospholipase A₂ assay method (Fluorescence-based phospholipase assays—Table 17.2) that is conceptually similar to the detection mechanism of ADIFAB. See below for more information on the ADIFAB fatty acid indicator.

EnzChek Direct Phospholipase C Assay Kit

The EnzChek Direct Phospholipase C Assay Kit (E10215, E10216) provides a simple and robust microplate-based method for monitoring phosphatidylcholine-specific phospholipase C (PC-PLC) activity in purified enzyme preparations. PC-PLC plays a crucial role in many cell signaling pathways involved in apoptosis and cell survival, as well as in diseases as diverse as cancer and HIV. This assay uses a proprietary substrate (glycerophosphoethanolamine with a dye-labeled sn-2 acyl chain) to detect PC-PLC activity. Substrate cleavage by PC-PLC releases the dye-labeled diacylglycerol, which produces a positive fluorescence signal that can be measured continuously using a fluorescence microplate reader. The reaction product has fluorescence excitation and emission maxima of 509 nm and 516 nm, respectively.

The EnzChek Direct Phospholipase C Assay Kit has been optimized using purified PC-PLC from Bacillus cereus. This assay may be amenable for use with cells and cell lysates, although the presence of phospholipase A₂ or phospholipase D activity can potentially result in signal enhancement. Using the EnzChek Direct Phospholipase C Assay Kit with purified enzyme from Bacillus cereus, we can detect as little as 10 mU/mL PC-PLC after one hour incubation at room temperature (Figure 17.47). This kit is also useful for characterizing PC-PLC inhibition, and because it offers a direct measurement, the potential for false positives in a compound screen is reduced.

Each EnzChek Direct Phospholipase C Assay Kit (2-plate size, E10215; 10-plate size, E10216) provides:

• Phosphatidylcholine-specific phospholipase C (PC-PLC) substrate
• Phospholipase C from Bacillus cereus
• Concentrated phospholipase C reaction buffer
• Phosphatidylcholine (lecithin)
• Dimethylsulfoxide (DMSO)
• Detailed assay protocols (EnzChek Direct Phospholipase C Assay Kit)

The 2-plate assay kit provides sufficient reagents for 200 reactions in 96-well microplates at a volume of 200 µL per well or 2000 reactions using low-volume 384-well microplates at a volume of 20 µL per well. The 10-plate assay kit provides sufficient reagents for 1000 reactions in 96-well microplates at a volume of 200 µL per well or 10,000 reactions using low-volume 384-well microplates at a volume of 20 µL per well.

Figure 17.47 Detection of phosphatidylcholine-specific phospholipase C (PC-PLC) using the EnzChek Direct Phospholipase C Assay Kit (E10215, E10216). Triplicate samples of PC-PLC from Bacillus cereus were assayed at concentration of 7.8 mU/mL to 500 mU/mL per well in the presence of 1X PLC substrate and 200 μM lecithin in 1X PLC reaction buffer. Reactions were incubated at room temperature for 60 minutes and fluorescence was measured using excitation/emission wavelengths of 490/520 nm. The inset represents a separate experiment and illustrates the linearity of fluorescence response at low levels of PC-PLC. The average variation of replicates (CV) was less than 3%. Background fluorescence determined for the no-enzyme control reaction has been subtracted.

Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit

The Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit (A12218) provides a sensitive method for continuously monitoring phosphatidylcholine-specific phospholipase C (PC-PLC) activity in vitro using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, PC-PLC activity is monitored indirectly using the Amplex Red reagent, a sensitive fluorogenic probe for H₂O₂ (Substrates for Oxidases, Including Amplex Red Kits—Section 10.5). First, PC-PLC converts the phosphatidylcholine (lecithin) substrate to form phosphocholine and diacylglycerol. After the action of alkaline phosphatase, which hydrolyzes phosphocholine to inorganic phosphate and choline, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, H₂O₂, in the presence of horseradish peroxidase, reacts with the Amplex Red reagent in a 1:1 stoichiometry to generate the highly fluorescent product, resorufin. Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

The Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit is potentially useful for detecting PC-PLC activity in cell extracts and for screening PC-PLC inhibitors. Experiments with purified PC-PLC from Bacillus cereus indicate that the Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit can detect PC-PLC levels as low as 0.2 mU/mL using a reaction time of one hour (Figure 17.30). One unit of PC-PLC is defined as the amount of enzyme that will liberate 1.0 micromole of water-soluble organic phosphorus from L-α-phosphatidylcholine per minute at pH 7.3 at 37°C.

Each Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit includes:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• Alkaline phosphatase from calf intestine
• L-α-Phosphatidylcholine (lecithin)
• Phosphatidylcholine-specific phospholipase C from Bacillus cereus
• Detailed protocols (Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay.

Figure 17.30 Detection of phosphatidylcholine-specific phospholipase C using the Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit (A12218). Fluorescence was measured in a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the sensitivity at very low enzyme concentrations.

Bacillus cereus PI-PLC

Phosphatidylinositol-specific phospholipase C (PI-PLC, EC 3.1.4.10) from Bacillus cereus cleaves phosphatidylinositol (PI), yielding water-soluble D-myo-inositol 1,2-cyclic monophosphate and lipid-soluble diacylglycerol. This enzyme also functions to release enzymes that are linked to glycosyl phosphatidylinositol (GPI) membrane anchors. Molecular Probes offers highly purified B. cereus PI-PLC (P6466), which has been used in studies of PI synthesis and export across the plasma membrane. PI-PLC generates diacylglycerols for PKC-linked signal transduction studies and provides an efficient means of releasing most GPI-anchored proteins from cell surfaces under conditions in which the cells remain viable.

Amplex Red Phospholipase D Assay Kit

The Amplex Red Phospholipase D Assay Kit (A12219) provides a sensitive method for measuring phospholipase D (PLD) activity in vitro using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, PLD activity is monitored indirectly using the Amplex Red reagent (Substrates for Oxidases, Including Amplex Red Kits—Section 10.5). First, PLD cleaves the phosphatidylcholine (lecithin) substrate to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, H₂O₂, in the presence of horseradish peroxidase, reacts with the Amplex Red reagent to generate the highly fluorescent product, resorufin (excitation/emission maxima ~571/585 nm).

The Amplex Red Phospholipase D Assay Kit is designed for detecting PLD activity in cell extracts and for screening PLD inhibitors.This kit can be used to continuously assay PLD enzymes with near-neutral pH optima, whereas PLD enzymes with acidic pH optima can be assayed in a simple two-step procedure. Experiments with purified PLD from Streptomyces chromofuscus indicate that the Amplex Red Phospholipase D Assay Kit can detect PLD levels as low as 10 mU/mL using a reaction time of one hour (Figure 17.32). One unit of PLD is defined as the amount of enzyme that will liberate 1.0 µmole of choline from L-α-phosphatidylcholine per minute at pH 8.0 at 30°C. Each Amplex Red Phospholipase D Assay Kit includes:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• L-α-Phosphatidylcholine (lecithin)
• Detailed protocols (Amplex Red Phospholipase D Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay.

Figure 17.32 Quantitation of phospholipase D from Streptomyces chromofuscus using the Amplex Red Phospholipase D Assay Kit (A12219). Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. The inset shows the sensitivity at very low enzyme concentrations (0–25 mU/mL).

Fluorescent Substrates for Phospholipase D

The products of phospholipase A₂, C and D cleavage of 1-O-alkyl-2-decanoyl-sn-glycero-3-phosphocholine labeled with the BODIPY FL fluorophore (D3771, ) can be separated and independently quantitated based on their differential migration on TLC or HPLC. Our BODIPY FL analog is preferred for this application because it is relatively photostable and the fluorescence properties of its different enzymatic products are all very similar. Researchers have taken advantage of these features to detect and quantitate phospholipase D activity in vascular smooth muscle cells, cultured mammalian cells and yeast.

The triacylglycerol-based EnzChek lipase substrate (E33955, ) offers higher throughput and better sensitivity than chromogenic (TLC or HPLC) assays, and a visible wavelength–detection alternative to pyrene-based fluorescent substrates. In the presence of lipases, the nonfluorescent EnzChek lipase substrate produces a bright, green-fluorescent product (excitation/emission maxima of ~505/515 nm) for the accurate and sensitive detection of lipase activity in solution. Furthermore, the green-fluorescent product of the EnzChek lipase substrate exhibits pH-insensitive spectra in the physiological pH range and is compatible with optics used for fluorescein detection in fluorometers.

Phosphatidylinositol (PI or PtdIns) and its phosphorylated derivatives represent only a small fraction of eukaryotic cellular phospholipids but are functionally significant in a disproportionately large number of regulatory and signal transduction processes. The most familiar of these processes is the phospholipase C–mediated generation of the ubiquitous second messengers inositol 1,4,5-triphosphate (InsP₃) and diacylglycerol (DAG) from phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P₂; Calcium Regulation—Section 17.2). Because these events are transient and spatially compartmentalized, methods for visualizing changes in the cellular localization of phosphoinositides are essential for their complete characterization. In collaboration with Echelon Biosciences, Inc. (http://www.echelon-inc.com), we offer biologically active fluorescent phosphoinositides, anti-phosphoinositide antibodies and immobilized phosphoinositide arrays.

Fluorescent Phosphoinositides

We offer fluorescent BODIPY analogs of the 3,4-phosphorylated and the 4,5-phosphorylated derivatives of PtdIns ( B34353, B34354; BODIPY dye–labeled phosphoinositides—Table 17.3). These fluorescent analogs are labeled with a BODIPY dye on the sn-1 acyl chain (Figure 17.33). This labeling pattern maximizes the separation of the dye from the inositol ring and ensures retention of biological activity. The phosphoinositide analogs can be used in combination with fluorescent organelle markers (Probes for Organelles—Chapter 12), calcium indicators (Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19) or GFP chimeras. When compared with alternative methods based on GFP–pleckstrin homology domain chimeras, detection of intracellular phosphoinositides using these probes is not complicated by parallel detection of soluble inositol phosphates and is subject to less distributional bias. A sensitive method for analyzing these fluorescent phosphoinositides and other products of lipid-modifying enzymes has been developed using a microfluidic chip device that separates lipids based on micellar electrokinetic capillary chromatography. Phosphoinositides can be delivered into live cells using polyamine reagents and detected with anti-phosphoinositide monoclonal antibodies (A21327, A21328; see below).

Anti-Phosphoinositide Monoclonal Antibodies

Research has revealed the direct action of phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P₂) and phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P₃) on a diverse array of cellular functions, including actin assembly and cytoskeletal dynamics, vesicular protein trafficking, protein kinase localization and activation, cell proliferation and apoptosis. We offer mouse monoclonal IgM antibodies to PtdIns(4,5)P₂ (A21327) and PtdIns(3,4,5)P₃ (A21328) for immunocytochemical localization of these important lipid metabolites (). Both antibodies have been shown to recognize their cognate phosphoinositides in murine and human cells with only slight crossreactivity with other phosphoinositides or phospholipids.

Arrays for Detecting Phosphoinositide–Protein Interactions

Protein domains that specifically bind phosphoinositides have emerged as major determinants in localizing proteins to their site of function. These phosphoinositide-binding motifs—which include the C2 (PKC conserved region 2), PH (pleckstrin homology), FYVE (Fab1p/YOTP/Vac1p/EEA1), ENTH (epsin NH₂-terminal homology) and PX (Phox homology) domains—are found in proteins implicated in a diverse array of cellular processes, such as actin cytoskeletal organization, cell growth regulation, control of gene expression, protein transport, exocytosis and endocytosis. Through localized phosphoinositide biosynthesis within the cell, proteins containing these lipid-recognition domains can be directed to functionally appropriate sites. PIP Strips and PIP Arrays, developed by Echelon Biosciences, Inc., are designed for identification of proteins possessing phosphoinositide recognition domains and analysis of their lipid-binding specificities.

PIP Strips provide 100 picomole samples of 15 different phospholipids, and a blank sample, immobilized on nitrocellulose membranes (Figure 17.37). We offer PIP Strips in packages of two (P23750) or ten (P23751) strips. PIP Strips are also available in a miniaturized format (PIP MicroStrips, P23752), which have been designed to allow immersion inside a standard microcentrifuge tube. PIP Arrays provide eight different phospholipids arrayed in amounts from 100 to 1.6 picomoles (Figure 17.38), allowing assessment of the strength of a protein's binding, in addition to its lipid specificity. We offer PIP Arrays in packages of two (P23748) or five (P23749) arrays. Phosphoinositide-mediated binding of proteins to PIP Strips and PIP Arrays is typically analyzed by protein–lipid overlay assays. Proteins may be detected using standard Western blot procedures in conjunction with our high-performance alkaline phosphatase– and horseradish peroxidase–mediated signal generation systems (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots—Section 9.4).

Sphingolipids include sphingomyelins, which are phospholipid analogs, as well as ceramides, glycosyl ceramides (cerebrosides), gangliosides and other derivatives (Figure 13.3). Several excellent reviews of the chemistry and biology of sphingolipids and glycosphingolipids and their role in the process of signal transduction are available.

BODIPY Sphingolipids

Ceramides (N-acylsphingosines), like diacylglycerols, are lipid second messengers that function in signal transduction processes. The concentration-dependent spectral properties of BODIPY FL C₅-ceramide (D3521, B22650; ), BODIPY FL C₅-sphingomyelin (D3522, ) and BODIPY FL C₁₂-sphingomyelin (D7711) make them particularly suitable for investigating sphingolipid transport and metabolism, in addition to their applications as structural markers for the Golgi complex (Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4, ). BODIPY FL C₅-ceramide can be visualized by fluorescence microscopy (, , ) or by electron microscopy following diaminobenzidine (DAB) photoconversion to an electron-dense product. (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).

Our range of BODIPY sphingolipids also includes the long-wavelength light–excitable BODIPY TR ceramide (D7540, ), as well as BODIPY FL C₅-lactosylceramide (D13951), BODIPY FL C₅-ganglioside G_M1 (B13950, ) and BODIPY FL C₁₂-galactocerebroside (D7519). All of Molecular Probes' sphingolipids are prepared from D-erythro-sphingosine and therefore have the same stereochemical conformation as natural biologically active sphingolipids.

Complexing fluorescent lipids with defatted bovine serum albumin (BSA) facilitates cell labeling by eliminating the need for organic solvents to dissolve the lipophilic probe; the BSA-complexed probe can be directly dissolved in water. We offer four BODIPY sphingolipid–BSA complexes for the study of lipid metabolism and trafficking, including:

• BODIPY FL C₅-ceramide (B22650)
• BODIPY TR C₅-ceramide (B34400)
• BODIPY FL C₅-lactosylceramide (B34401)
• BODIPY FL C₅-ganglioside G_M1 (B34402)

BODIPY FL C₅-ceramide has been used to investigate the linkage of sphingolipid metabolism to protein secretory pathways and neuronal growth. Internalization of BODIPY FL C₅-sphingomyelin (D3522) from the plasma membrane of human skin fibroblasts results in a mixed population of labeled endosomes that can be distinguished based on the concentration-dependent green (~515 nm) or red (~620 nm) emission of the probe (). BODIPY C₅-sphingomyelin has also been used to assess sphingomyelinase gene transfer and expression in hematopoietic stem and progenitor cells. BODIPY FL C₅-lactosylceramide, BODIPY FL C₅-ganglioside G_M1 and BODIPY FL cerebrosides should be useful tools for the study of glycosphingolipid transport and signaling pathways in cells and for diagnosis of lipid-storage disorders such as Niemann–Pick disease, Gaucher disease, G_M1 gangliosidosis, Morquio syndrome and type IV mucolipidosis (ML-IV). Addition of BODIPY FL C₅-lactosylceramide to the culture medium of cells from patients with sphingolipid-storage diseases (sphingolipidosis) results in fluorescent product accumulation in lysosomes, whereas this probe accumulates in the Golgi apparatus of normal cells and cells from patients with other storage diseases. BODIPY FL C₅-ganglioside G_M1 has been shown to form cholesterol-enhanced clusters in membrane complexes with amyloid β-protein in a model of Alzheimer disease amyloid fibrils. Colocalization of fluorescent cholera toxin B conjugates (Lectins and Other Carbohydrate-Binding Proteins—Section 7.7) and BODIPY FL C₅-ganglioside G_M1 observed by fluorescence microscopy provides a direct indication of the association of these molecules in lipid rafts (, ). Studies by Martin and Pagano have shown that the internalization routes for BODIPY FL C₅-glucocerebroside follow both endocytic and nonendocytic pathways and are quite different from those for BODIPY FL C₅-sphingomyelin.

NBD Sphingolipids

NBD C₆-ceramide (N1154, ) and NBD C₆-sphingomyelin (N3524) analogs predate their BODIPY counterparts and have been extensively used for following sphingolipid metabolism in cells and in multicellular organisms. As with BODIPY FL C₅-ceramide, we also offer NBD C₆-ceramide complexed with defatted BSA (N22651) to facilitate cell loading without the use of organic solvents to dissolve the probe. Koval and Pagano have prepared NBD analogs of both the naturally occurring D-erythro and the nonnatural L-threo stereoisomers of sphingomyelin and have compared their intracellular transport behavior in Chinese hamster ovary (CHO) fibroblasts. NBD C₆-ceramide lacks the useful concentration-dependent optical properties of the BODIPY FL analog and is less photostable; however, the fluorescence of NBD C₆-ceramide is apparently sensitive to the cholesterol content of the Golgi apparatus, a phenomenon that is not observed with BODIPY FL C₅-ceramide. If NBD C₆-ceramide–containing cells are starved for cholesterol, the NBD C₆-ceramide that accumulates within the Golgi apparatus appears to be severely photolabile, but this NBD photobleaching can be reduced by stimulation of cholesterol synthesis. Thus, NBD C₆-ceramide may be useful in monitoring the cholesterol content of the Golgi apparatus in live cells.

Arrays for Detection of Sphingolipid–Protein Interactions

Sphingolipid–protein interactions are involved in physiological and pathological processes, including signal transduction mediated by G-protein–coupled receptors (GPCRs), induction of apoptosis and amyloid fibril formation. SphingoStrips, developed by Echelon Biosciences, Inc., are designed for identification of proteins possessing sphingolipid recognition domains and analysis of their lipid-binding specificities. SphingoStrips provide 100 picomole samples of 15 different lipids, and a blank sample, immobilized on nitrocellulose membranes (Figure 17.37), and are offered in packages of 10 strips (S23753). Membrane-bound proteins may be detected using standard Western blot procedures in conjunction with our high-performance alkaline phosphatase– and horseradish peroxidase–mediated signal generation systems (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots—Section 9.4).

Vybrant Lipid Raft Labeling Kits

The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405) are designed to provide convenient, reliable and extremely bright fluorescent labeling of lipid rafts in live cells. Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by their selective interactions with these membrane microdomains. Recent research has demonstrated that lipid rafts play a role in a variety of cellular processes—including the compartmentalization of cell-signaling events, the regulation of apoptosis and the intracellular trafficking of certain membrane proteins and lipids —as well as in the infectious cycles of several viruses and bacterial pathogens. Examining the formation and regulation of lipid rafts is a critical step in understanding these aspects of eukaryotic cell function.

The Vybrant Lipid Raft Labeling Kits provide the key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes (, ). Live cells are first labeled with the green-fluorescent Alexa Fluor 488, orange-fluorescent Alexa Fluor 555 or red-fluorescent Alexa Fluor 594 conjugate of cholera toxin subunit B (CT-B). This CT-B conjugate binds to the pentasaccharide chain of plasma membrane ganglioside G_M1, which selectively partitions into lipid rafts. All of Molecular Probes' CT-B conjugates are prepared from recombinant CT-B and are completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. An antibody that specifically recognizes CT-B is then used to crosslink the CT-B–labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by fluorescence microscopy.

Each Vybrant Lipid Raft Labeling Kit contains sufficient reagents to label 50 live-cell samples in a 2 mL assay, including:

• Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor 488 (in Kit V34403), Alexa Fluor 555 (in Kit V34404) or Alexa Fluor 594 (in Kit V34405) dye
• Anti–cholera toxin subunit B antibody (anti–CT-B)
• Concentrated phosphate-buffered saline (PBS)
• Detailed labeling protocols (Vybrant Lipid Raft Labeling Kits)

Because they are compatible with various multilabeling schemes, the Vybrant Lipid Raft Labeling Kits can also serve as important tools for identifying physiologically significant membrane proteins that associate with lipid rafts. Cells can be labeled with other live-cell probes during the lipid raft labeling protocol or immediately following the antibody crosslinking step, depending on the specific labeling requirements of the other probes. Alternatively, once the lipid rafts have been labeled and crosslinked, the cells can be fixed for long-term storage or fixed and permeabilized for subsequent labeling with antibodies or other probes that are impermeant to live cells.

Amplex Red Sphingomyelinase Assay Kit

The Amplex Red Sphingomyelinase Assay Kit (A12220) is designed for measuring sphingomyelinase activity in solution using a fluorescence microplate reader or fluorometer (Figure 13.35). This assay should be useful for screening sphingomyelinase activators or inhibitors or for detecting sphingomyelinase activity in cell and tissue extracts. The assay, which uses natural sphingomyelin as the principal substrate, employs an enzyme-coupled detection scheme in which phosphocholine liberated by the action of sphingomyelinase is cleaved by alkaline phosphatase to generate choline. Choline is, in turn, oxidized to betaine by choline oxidase, generating H₂O₂, which drives the conversion of the Amplex Red reagent (A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits—Section 10.5) to red-fluorescent resorufin (). This sensitive assay technique has been employed to detect activation of acid sphingomyelinase associated with ultraviolet radiation–induced apoptosis and to characterize an insecticidal sphingomyelinase C produced by Bacillus cereus.

The Amplex Red Sphingomyelinase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• Alkaline phosphatase from calf intestine
• Sphingomyelin
• Triton X-100
• Sphingomyelinase from Staphylococcus sp.
• Detailed protocols (Amplex Red Sphingomyelinase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay.

Figure 13.35 Measurement of sphingomyelinase activity using the Amplex Red Sphingomyelinase Assay Kit (A12220). Each reaction contained 50 µM Amplex Red reagent, 1 U/mL horseradish peroxidase (HRP), 0.1 U/mL choline oxidase, 4 U/mL of alkaline phosphatase, 0.25 mM sphingomyelin and the indicated amount of Staphylococcus aureus sphingomyelinase in 1X reaction buffer. Reactions were incubated at 37°C for one hour. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

Elevated levels of free fatty acids (FFA)—which are associated with multiple pathological states, including cancer, diabetes and cardiac ischemia —are generated by inflammatory responses, phospholipase A activity and cytotoxic phenomena. Sensitive techniques are required to detect and quantitate free fatty acids because these important metabolites have low aqueous solubility and are usually found complexed to carriers. ADIFAB (A3880) is a dual-wavelength fluorescent FFA indicator that consists of a polarity-sensitive fluorescent probe (acrylodan, A433; Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3) conjugated to I-FABP, a rat intestinal fatty acid–binding protein with a low molecular weight (15,000 daltons) and a high binding affinity for FFA (Figure 17.42).

As shown in Figure 17.43, titration of the ADIFAB reagent with oleic acid results in a shift of its fluorescence maximum from ~432 nm to ~505 nm. The ratio (R) of these signals (505 nm/432 nm) can be converted to an FFA concentration by using the FFA dissociation constant (K_d) and employing analysis procedures similar to those developed for Ca²⁺ indicators (Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19). Values of K_d vary considerably for different fatty acids; a typical value is 0.28 µM for oleic acid (determined at 37°C). There is little, if any, interference from bile acids, glycerides, sterols or bilirubin. With appropriate precautions, which are described in the product information sheet accompanying this product (ADIFAB Free Fatty Acid Indicator), ADIFAB can be used to determine FFA concentrations in the range 1 nM to >20 µM.

ADIFAB was used to investigate the physical basis of cis-unsaturated fatty acid inhibition of cytotoxic T cells. This effect is due to inhibition of a specific tyrosine phosphorylation event that normally accompanies antigen stimulation. Measurements using ADIFAB have also revealed previously undetected differences in FFA binding affinities among fatty acid–binding proteins from different tissues and have enabled quantitation of FFA levels in human serum as a potential diagnostic tool.

Figure 17.43 The free fatty acid–dependent spectral shift of ADIFAB (A3880). Spectra shown represent 0.2 µM ADIFAB in pH 8.0 buffer with (+OA) and without (–OA) addition of 4.7 µM cis-9-octadecenoic (oleic) acid (OA). The ratio of fluorescence emission intensities at 505 nm and 432 nm can be quantitatively related to free fatty acid concentrations.