Genetic analysis of digestive physiology using fluorescent phospholipid reporters.

Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

Efficient synthesis of the cholinephosphate phospholipid headgroup.

In search of an efficient method to prepare cholinephosphate headgroups in phospholipids under mild conditions (where the diacylglycerol moiety is not subject to oxidation), a method was developed for phosphorylation using a trialkyl phosphite and I2. The active intermediate is a phosphoryl iodide formed by oxidation of the phosphite with I2. 2-Bromoethanol, dimethyl chlorophosphite, and an alcohol (diglyceride) are converted to a phosphate triester in a one-pot reaction with high yield. In the second reaction, the phosphate triester is demethylated, and the ethyl bromide group is converted to choline by treatment with aqueous trimethylamine. This procedure is applied to the synthesis of hexadecylphosphocholine, and 1,2-didecanoyl-1-deoxy-1-thio-sn-glyceryo-3-phosphocholine.

Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging.

Phospholipase substrate analogs containing both a fluorescent BODIPY group and a quenching 2,4-dinitrophenyl (DNP) group were synthesized. They showed little fluorescence, but upon hydrolysis became fluorescent as the quenching group was removed. Two substrates were phosphatidylethanolamine analogs with a BODIPY-pentanoyl group at the sn-2 position and DNP linked to the amino head group. The third was a phosphatidylcholine analog with a BODIPY-labeled alkyl ether at the sn-1 position and a N-(DNP)-8-amino-octanoyl group at the sn-2 position. These compounds were evaluated as substrates for cytosolic (85 kDa) phospholipase A(2) (cPLA(2)) and plasma platelet-activating factor acetylhydrolase (rPAF-AH). Two were good substrates for cPLA(2) (specific activities: 18 and 5 nmol min(-1) mg(-1)) and all were good for rPAF-AH (specific activities: 17, 11, and 6 micro mol min(-1) mg(-1)). The minimal amount of enzyme detectable was 50 ng for cPLA(2) and 0.1 ng for rPAF-AH. These substrates were active in assays of PLA(2) in zebrafish embryo extracts and one was well suited for imaging of PLA(2) activity in living zebrafish embryos. Embryos were injected with substrate at the one- to four-cell stage and allowed to develop until early somitogenesis when endogenous PLA(2) activity increases dramatically; substrate persisted (12 h) and specifically labeled cells of the developing notochord.

Binding of phosphatidylinositol-specific phospholipase C to phospholipid interfaces, determined by fluorescence resonance energy transfer.

Dissociation constants for binding of phosphatidylinositol-specific phospholipase C from Bacillus cereus (bcPI-PLC) and the mammalian phosphatidylinositol-specific phospholipase C-delta(1) to lipid interfaces containing phosphoinositol, phosphocholine, and phosphomethanol head groups were determined by fluorescence resonance energy transfer. Dansyl-labeled lipid probes were used as acceptors, with intrinsic tryptophan of the enzyme as the donor. Titration of protein into lipid provided data from which K(d) and N, the limiting number of lipid molecules per protein bound, were calculated by non-linear regression analysis of exact binding equations. These results were compared with apparent K(m) values from kinetic data. K(d) values in the low microM range in terms of lipid monomers or low nM range in terms of binding sites were calculated with good fits of experimental data to theoretical binding curves. bcPI-PLC binds with high affinity to PI interfaces, slightly lower affinity to PC interfaces, and much lower affinity to PM interfaces. The mammalian enzyme also binds with high affinity to PI interfaces, but shows little or no binding with PC interfaces under similar concentration conditions. These K(d) values correlate reasonably with apparent K(m) values from kinetic experiments.

A thiophosphate analog of dimyristoylphosphatidyl-inositol-4-phosphate is a substrate for mammalian phosphoinositide-specific phospholipase C.

1,2-Dimyristoyloxypropane-3-thiophosphate(rac-1-myo-inositol-4- phosphate), a thiophosphate analog of dimyristoyl phosphatidylinositol-4-phosphate was synthesized as a substrate for mammalian phosphoinositide-specific phospholipase C. Its activity with delta(1-132)-PI-PLC-delta 1 (a deletion mutant with the N-terminal pleckstrin homology domain removed) was studied in sonicated dispersions, with and without added Triton X-100. It had an initial activity of about 30 mumol min-1 mg-1, which rapidly decreased due to substrate depletion in the vesicle or micelle. The slower rate of hydrolysis appeared limited by enzyme hopping or exchange of substrate between vesicles or micelles, which was more rapid in the presence of detergent.

Kinetics of phosphatidylinositol-specific phospholipase C with vesicles of a thiophosphate analogue of phosphatidylinositol.

1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was synthesized as a substrate for the continuous spectrophotometric assay of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. Release of thio-diglyceride is followed by a coupled reaction with 4,4'-dithiopyridine to produce a chromophore, 4-thiopyridine, measured by its absorption at 324 nm. Sonicated vesicles of D-thio-DMPI gave sigmoidal Michaelis-Menten kinetics with PI-PLC as a function of bulk concentration of substrate (Hill plot: Vmax = 132 mumol min-1 mg-1, apparent Km = 0.115 mM, h = 1.8). Addition of dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylmethanol to vesicles of D-thio-DMPI resulted in an initial increase in rate followed by a decrease at higher concentrations of non-substrate lipid. Binding of PI-PLC to vesicles of DMPC with 10 mol% of N-dansyl phosphatidylethanolamine was demonstrated by fluorescence resonance energy transfer from tryptophan in the enzyme to the dansyl lipid probe.

Synthesis of optically-active hexadecyl thiophosphoryl-1-D-myo-inositol: a thiophosphate analog of phosphatidylinositol.

The synthesis of optically-active hexadecyl thiophosphoryl-1-D-myo-inositol 11 was accomplished from 2,3-O-(D-1',7',7'-trimethyl[2.2.1]bicyclohept-2'-ylidene)-4,5,6-O- tris(methoxymethyl)-D-myo-inositol 6 or 2,3,4,5,6-O-pentakis(methoxymethyl)-D-myo-inositol 14, using the Arbusov reaction of their dimethyl phosphite derivatives 7 and 15 with N-hexadecyl thiophthalimide 8. This product was a substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus.

Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol.

Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus. Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine. Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection. The activity of PI-PLC with thio-PI analogs showed an interfacial effect. C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC). Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC. All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration. The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1. With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.

Interfacial catalysis by phospholipase A2: the rate-limiting step for enzymatic turnover.

The kinetics of the phospholipase A2-catalyzed hydrolysis of bilayer vesicles and mixed micelles of several oxyglycero and thioglycero analogues of phospholipids have been studied. The results with vesicles show that, depending on the source of the enzyme, the rates of hydrolysis of the oxy-containing long-chain phosphatidylmethanols are 2.5- to 28-fold higher compared to the rates of hydrolysis of the analogous thio substrates. The oxygen to sulfur substitution does not significantly alter the affinities of the enzymes for the reaction products or calcium. Since it is unlikely that sulfur substitution changes the rate constants for the formation and dissociation of the enzyme-product complex by the same factor, the element effects seen in the rates of hydrolysis of the oxy- and thioester phospholipids in vesicles are primarily due to a change in the rate constant for the chemical step of the catalytic turnover cycle. For bovine pancreatic phospholipase A2, various mutants with lower catalytic activity were used to show that the value of the element effect does not increase in the mutants. These results establish that, for the pancreatic phospholipase A2, the element effect is fully expressed, and the chemical step is fully rate-limiting for both oxyglycero and thioglycero phospholipids in vesicles. It was found that the element effect decreases from 7 to 1 when long-chain phosphatidylmethanols are present in micelles of a neutral diluent. This result suggests that the chemical step is not rate-limiting during the hydrolysis of these mixed micelle substrates.

A facile asymmetric synthesis of glycerol phospholipids via tritylglycidol prepared by the asymmetric epoxidation of allyl alcohol. Thiolester and thioether analogs of phosphatidylcholine.

Trityl-glycidol was synthesized by in situ derivatization of glycidol, which was prepared by the catalytic asymmetric epoxidation of allyl alcohol. Depending on the enantiomer of diisopropyl tartrate used with the titanium catalyst, either (R)- or (S)-trityl-glycidol was obtained in a "one pot" synthesis in about 50% overall yield. The optical purity, determined by NMR spectroscopy of a Mosher ester, was greater than 98% ee. Nucleophilic opening of the chiral epoxide with dodecyl mercaptan gave optically active 1-S-dodecyl-3-O-trityl-1-thio-glycerol, which was used to synthesize 1-S-dodecyl-2-O-decanoyl-thio-sn-glycero-3-phosphocholine. Opening of the epoxide with methyl xanthate gave a 1,2-trithiocarbonate derivative of trityl glycerol which can be used to synthesize 1,2-bis(S-decanoyl)-1,2-dithio-sn-glycero-3-phosphocholine. Opening of the epoxide with thiodecanoic acid gave 1-S-decanoyl-3-O-trityl-1-thio-glycerol which was used to synthesize 1-S-decanoyl-2-O-decanoyl-1-thio-sn-glycero-3-phosphocholine.

Evaluation of fluorescent and colored phosphatidylcholine analogs as substrates for the assay of phospholipase A2.

Two fluorescent phospholipid analogs, 1-O-[12-(2-naphthyl)-dodec-11-enyl]-2-O-decanoyl-sn-glycero-3-phos phocholine and dansyl-PC [rac 1-O-(N-dansyl-11- amino-1-undecyl)-2-O-decanoyl-glycero-3-phosphocholine], and a red-colored analog, dabsyl-PC [rac 1-O-(N-dabsyl-11-amino-1-undecyl)-2-O-decanoyl-glycero-3- phosphocholine], were evaluated as substrates for the assay of phospholipase A2 (PLA2) (Crotalus adamanteus). The assay reaction was monitored by separation of the fluorescent or colored substrate and product by TLC and quantitation by fluorescence or absorption scanning. All three substrates gave similar (within an order of magnitude) activities with PLA2. Dansyl-PC was best suited to this TLC assay. Dabsyl-PC was less sensitive to detection by absorbance, but had the advantage of being red colored and readily detected by the unaided eye.

Synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine and its use in the assay of phospholipase A2.

The synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine, 1-O-[12-(2-naphthyl)-dodec-11-enyl]-2-O-decanoyl-sn-glycerol-3- phosphocholine (NVPC), is described. This involves a Wittig reaction between 2-naphthaldehyde and a phosphonium salt which gives the trans-naphthylvinyl group as the predominant isomer. Lyso NVPC was prepared from NVPC by phospholipase A2 action. NVPC absorbs strongly at 248 nm (epsilon = 58,300 M-1 cm-1) and gives broad fluorescence emission with maxima at 343 nm and 360 nm and a quantum yield of 0.10 in ethanol. An assay for phospholipase A2 was developed using high performance liquid chromatography with fluorescence detection to separate and quantify NVPC and lyso NVPC. Activities as low as 1-2 pmol/min in an assay volume of 0.1 ml can easily be measured. The assay was used with a pure enzyme from cobra venom and a crude enzyme from synovial fluid. Enzyme specificities for phosphatidylcholine and NVPC with cobra venom and porcine pancreatic phospholipases A2 were compared using a titrametric assay. The use of the assay with NVPC to study the metabolism of platelet activating factor is discussed.

Chiral synthesis of a dithiolester analog of phosphatidylcholine as a substrate for the assay of phospholipase A2.

The synthesis of a dithiolester analog of phosphatidylcholine, 1,2-bis(heptanoylthio)-1,2-dideoxy-sn-glycerol-3-phosphocholine (thio PC), is described. Starting with 1-trityl-sn-glycerol (prepared from D-mannitol), tosylation followed by displacement with potassium methyl xanthate gave a trithiocarbonate. Reductive cleavage of the latter gave a 1,2-dithiol which was then acylated, detritylated, and esterified with choline phosphate. Hydrolysis of thio PC by phospholipase A2 (Naja naja) indicated 95% chiral purity. The rate of hydrolysis as a function of substrate concentration showed a sharp increase at about 0.17 mM, the critical micellar concentration of the lipid. A spectrophotometric assay of phospholipase A2 (by measurement of released thiol groups in the presence of dithionitrobenzoic acid) was quite sensitive. As little as 1 ng of enzyme was detected, representing a rate of about 0.2 nmol of substrate hydrolyzed per min.