Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1ß, and interleukin 1 receptor antagonist but not transforming growth factor ß1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents.

Comprehensive portrait of cholesterol containing oxidized membrane.

Biological membranes are under significant oxidative stress caused by reactive oxygen species mostly originating during cellular respiration. Double bonds of the unsaturated lipids are most prone to oxidation, which might lead to shortening of the oxidized chain and inserting of terminal either aldehyde or carboxylic group. Structural rearrangement of oxidized lipids, addressed already, is mainly associated with looping back of the hydrophilic terminal group. This contribution utilizing dual-focus fluorescence correlation spectroscopy and electron paramagnetic resonance as well as atomistic molecular dynamics simulations focuses on the overall changes of the membrane structural and dynamical properties once it becomes oxidized. Particularly, attention is paid to cholesterol rearrangement in the oxidized membrane revealing its preferable interaction with carbonyls of the oxidized chains. In this view cholesterol seems to have a tendency to repair, rather than condense, the bilayer.

Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

Conformations of double-headed, triple-tailed phospholipid oxidation lipid products in model membranes.

Products of phospholipid oxidation can produce lipids with a carbonyl moiety at the end of a shortened lipid acyl tail, such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The carbonyl tail of POVPC can covalently bond to the free tertiary amine of a phosphatidylethanolamine lipid in a Schiff base reaction to form a conjugate lipid (SCH) with two head groups, and three acyl tails. We investigate the conformations and properties of this unique class of adduct lipids using molecular dynamics simulations, and show that their insertion into lipid bilayers of POPC increases the average cross-sectional area per lipid and decreases bilayer thickness. Significant increase in acyl tail fluidity is only observed at 25% SCH concentration. The SCH occupies a larger area per lipid than expected for a lipid with three acyl tails, owing to the interfacial location of the long spacer between the two head groups of the SCH. Schiff base formation of lipids can alter the concentration, homeostasis and localizations of phosphatidylserine and phosphatidylethanol lipids in membranes, and can therefore influence several membrane-associated processes including fusion and budding. The current work provides the first detailed structural model of this unique new class of lipids that may have important roles to play in modulating membrane properties and cell physiology.

Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons.

Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4°C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55-67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) deficiencies affect expression of lipolytic activities in mouse adipose tissues.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patterns of brown and white adipose tissue from ATGL (-/-) and HSL (-/-) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols.

Protein modification by aldehydophospholipids and its functional consequences.

Phospholipid aldehydes represent a particular subclass of lipid oxidation products. They are chemically reactive and can form Schiff bases with proteins and aminophospholipids. As chemically bound molecular entities they modulate the functional properties of biomolecules in solution and the surface of supramolecular systems including plasma lipoproteins and cell membranes. The lipid-protein and lipid-lipid conjugates may be considered the active primary platforms that are responsible for the biological effects of aldehydophospholipids, e.g. receptor binding, cell signaling, and recognition by the immune system. Despite the fact that aldehydophospholipids are covalently associated, they are subject to exchange between nucleophiles since their imine conjugates are not stable. As a consequence, aldehydophospholipids exist in a dynamic equilibrium between different "states" depending on the lipid and protein environment. Aldehydophospholipids may also contribute to the systemic administration and activity of oxidized phospholipids by inducing release of microparticles by cells. These effects are lipid-specific. Future studies should help clarify the mechanisms and consequences of these membrane-associated effects of "phospholipid stress". This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae.

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.

Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes.

Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes.

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds.

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.

Toxicity of oxidized phospholipids in cultured macrophages.

BACKGROUND: The interactions of oxidized low-density lipoprotein (LDL) and macrophages are hallmarks in the development of atherosclerosis. The biological activities of the modified particle in these cells are due to the content of lipid oxidation products and apolipoprotein modification by oxidized phospholipids. RESULTS: It was the aim of this study to determine the role of short-chain oxidized phospholipids as components of modified LDL in cultured macrophages. For this purpose we investigated the effects of the following oxidized phospholipids on cell viability and apoptosis: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and oxidized alkylacyl phospholipids including 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine (E-PGPC) and 1-O-hexadecyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (E-POVPC). We found that these compounds induced apoptosis in RAW264.7 and bone marrow-derived macrophages. The sn-2 carboxyacyl lipid PGPC was more toxic than POVPC which carries a reactive aldehyde function in position sn-2 of glycerol. The alkylacyl phospholipids (E-PGPC and E-POVPC) and the respective diacyl analogs show similar activities. Apoptosis induced by POVPC and its alkylether derivative could be causally linked to the fast activation of an acid sphingomyelinase, generating the apoptotic second messenger ceramide. In contrast, PGPC and its ether analog only negligibly affected this enzyme pointing to an entirely different mechanism of lipid toxicity. The higher toxicity of PGPC is underscored by more efficient membrane blebbing from apoptotic cells. In addition, the protein pattern of PGPC-induced microparticles is different from the vesicles generated by POPVC. CONCLUSIONS: In summary, our data reveal that oxidized phospholipids induce apoptosis in cultured macrophages. The mechanism of lipid toxicity, however, largely depends on the structural features of the oxidized sn-2 chain.

Activity-based probes for studying the activity of flavin-dependent oxidases and for the protein target profiling of monoamine oxidase inhibitors.

High profile: new activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinson's disease, shows unique protein specificity despite its covalent mechanism of action.

Functional proteomic analysis of lipases and esterases in cultured human adipocytes.

This study reports on the analysis of the lipolytic proteome of cultured human fat cells. We used specific affinity tags to detect and identify the lipolytic and esterolytic enzymes in human subcutaneous (Sc) and visceral (Visc) adipocytes. For this purpose, differentiated fat cells were incubated with a fluorescent suicide inhibitor followed by protein separation using one- or two-dimensional gel electrophoresis. After detection by fluorescence laser scanning, the labeled proteins were tryptically digested and peptides were identified by mass spectrometry. In addition, a biotinylated probe was used for specific enzyme labeling with subsequent avidin affinity isolation of the tagged proteins. Finally, we determined the quantitative differences in protein expression levels between subcutaneous and visceral adipocytes using differential activity-based gel electrophoresis (DABGE). We found that the lipase/esterase patterns of both cell types are very similar, except for some proteins that were only found in Sc cells. Two novel enzyme candidates identified in this study were overexpressed and characterized using biologically relevant glycerolipid substrates in vitro. Both of them showed pronounced hydrolytic activities on hydrophobic acylglycerols and therefore may be considered lipases. The physiological functions of the novel lipolytic proteins in vivo are currently subject to investigation.

Development of a novel, cell-based chemical screen to identify inhibitors of intraphagosomal lipolysis in macrophages.

Macrophages play a central role in tissue homeostasis and the immune system. Their primary function is to internalize cellular debris and microorganisms for degradation within their phagosomes. In this context, their capacity to process and sequester lipids such as triacylglycerides and cholesteryl esters makes them key players in circulatory diseases, such as atheroclerosis. To discover new inhibitors of lipolytic processing within the phagosomal system of the macrophage, we have developed a novel, cell-based assay suitable for high-throughput screening. We employed particles carrying a fluorogenic triglyceride substrate and a calibration fluor to screen for inhibitors of phagosomal lipolysis. A panel of secondary assays were employed to discriminate between lipase inhibitors and compounds that perturbed general phagosomal trafficking events. This process enabled us to identify a new structural class of pyrazole-methanone compounds that directly inhibit lysosomal and lipoprotein lipase activity.

Addressable bipartite molecular hook (ABMH): immobilized hairpin probes with sensitivity below 50 fM.

Sensitivity and specificity of nucleic acid binding probes immobilized on solid supports are essential features of microarrays. Whereas conventional biochips apply nonquenched linear probes (cDNA, oligonucleotides), hairpin structures containing a fluorophore-quencher system comprise important prerequisites required for ideal transcriptional probes. We describe here the generation of addressable bipartite molecular hook (ABMH) probes and the characterization of their performance analyzing biological and clinical samples, also in comparison to linear oligonucleotide arrays. ABMH can be immobilized subsequent to reaction with the target sequence or the reaction carried out directly with the immobilized probe; target sequences are recognized with excellent sensitivity, specificity, and a detection limit below 50 fM. Due to excellent sensitivity and specificity, ABMH represent ideal candidates for the nonamplified microarray-based detection of low abundance nucleic acids, e.g., required in diagnostic assays.

Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer.

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane.

Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities.

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision.

Recording phagosome maturation through the real-time, spectrofluorometric measurement of hydrolytic activities.

The efficient degradation of internalized particulate matter is a principal objective of the macrophage's phagosome. Assessment of the true hydrolytic capacity within the phagosomal lumen is often difficult as it is subject to many factors beyond recruitment of lysosomal hydrolases. Here we outline three assays that allow quantitative measurements of serine-cysteine protease, triglyceride lipase, and beta-galactosidase activities within the phagosomes of macrophages, in real time. The assays utilize ratio fluorometry between particle-associated fluorogenic substrates and calibration fluorochromes to yield internally controlled values that record rates of substrate hydrolysis. The methods described utilize a spectrofluorometer for fluorometric measurements from a population of macrophages. These assays, however, can be expanded to high-throughput or single cell formats. In addition, this approach can be applied to measure a wide variety of phagosomal hydrolytic properties with the design of suitable fluorogenic substrates.