Ceramides modulate protein kinase C activity and perturb the structure of Phosphatidylcholine/Phosphatidylserine bilayers.

We studied the effects of natural ceramide and a series of ceramide analogs with different acyl chain lengths on the activity of rat brain protein kinase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC)/dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using (2)H-NMR and specific enzymatic assays in the absence or presence of 7.5 mol % diolein (DO). Only a slight activation of PKC was observed upon addition of the short-chain ceramide analogs (C(2)-, C(6)-, or C(8)-ceramide); natural ceramide or C(16)-ceramide had no effect. In the presence of 7.5 mol % DO, natural ceramide and C(16)-ceramide analog slightly attenuated DO-enhanced PKC activity. (2)H-NMR results demonstrated that natural ceramide and C(16)-ceramide induced lateral phase separation of gel-like and liquid crystalline domains in the bilayers; however, this type of membrane perturbation has no direct effect on PKC activity. The addition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C(6)-ceramide and 15 mol % DO. Further increases in C(6)-ceramide and/or DO concentrations led to decreased PKC activity. A detailed (2)H-NMR investigation of the combined effects of C(6)-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to adopt nonbilayer structures, resulting in the actual presence of such structures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the increased tendency to form nonbilayer lipid phases correlates with increased PKC activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analogs, widely used to study cellular effects of ceramide, have biological effects that are not exhibited by natural ceramide.     

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.     

Action of pancreatic phospholipase A2 on phosphatidylcholine bilayers in different physical states.

1. Saturated and unsaturated phosphatidylcholines, dispersed as liposomes in water, can be hydrolysed by phospholipase A2 from pig pancreas. A pure saturated phosphatidylcholine is hydrolysed only near its transition temperature. An unsaturated phosphatidylcholine is hydrolysed preferentially near its transition temperature, but hydrolysis can occur also above the transition temperature, albeit at a much lower rate. 2. An equimolar mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, which shows cocrystallization of the paraffin chains, is hydrolyzed between 25 and 40 degrees C with a maximum at 32 degrees C, in agreement with the calorimetric scan of the phase transition. 3. An equimolar mixture of dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine, which shows a monotectic behaviour, is hydrolysed at all temperatures. Hydrolysis is maximal at 0 and 40 degrees C, at which temperatures dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine undergo their phase transition, respectively. 4. Both in the mixture showing cocrystallization and in the mixture in which phase separation occurs, the phosphatidylcholine species with the shorter fatty acid chains is hydrolysed at a higher rate than the longer chain fatty acid species. 5. Hydrolysis is inhibited by the presence of cholesterol in liposomes prepared of saturated phosphatidylcholine. Inhibition is complete at a cholesterol concentration of 35 mol %. Subsequent addition of filipin and amphotericin B to the mixed cholesterol-phosphatidylcholine liposomes overcomes the inhibitory effect of cholesterol.     

华麻花头根中的神经酰胺成分

从菊科麻花头属植物华麻花头（Serratula chinensis S．Moore）根中分离得一组神经酰胺类化俞物,经光谱和化学方法分析,鉴定为（2S,3S,4R,8E）-8,9-二脱氢植物鞘氨醇（2R）-2-羟基脂肪酰胺,其脂肪酸链主要由十六,二十二,二十三,二十四和二十五烷酸组成,其中2-羟基十六碳酸组成的神经酰胺（2S,3S,4R,8E）-2-[（2R）-2-羟基棕榈酰胺]-8-十八碳烯-1,3,4-三醇为一新化合物。高锰酸钾氧化法被应用于判断单不饱和长链中双键的化置。     

The relationship between compositional phase separation and vesicle morphology: Implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A₂ (PLA₂) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA₂. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Action of phospholipase A2 on unmodified phosphatidylcholine bilayers: Organizational defects are preferred sites of action

Summary The hydrolytic action of the bee venom phospholipase A₂ on phosphatidylcholine bilayers is studied under a variety of conditions that introduce alterations in the packing, such as those induced by sonication, gel to liquid crystalline phase transition, and osmotic shock. Two phases of hydrolysis could be resolved under a wide range of experimental conditions. With the various forms of the bilayers one observes only a partial hydrolysis of the total available substrate during the first phase. However, the fraction of the substrate hydrolyzed in the first phase changes with the form of the available substrate, with the amount of the enzyme added, with the temperature, with the phase transition characteristics of the substrate, and by the sonication of the substrate. The second phase of hydrolysis is generally observed when a certain concentration of the products has been produced during the first phase of hydrolysis. These observations are interpreted to suggest that the bee venom phospholipase A₂ preferentially catalyzes hydrolysis of the substrate available at or near the defects in the organization of the substrate in the bilayers.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

Ceramides with 2-hydroxylated,very long-chain polyenoic fatty acids in rodents: From testis to fertilization-competent spermatozoa

Sphingolipids from rodent testis and spermatozoa are known to contain non-hydroxylated (N-) and 2-hydroxylated (2-OH) very-long-chain polyunsaturated fatty acids (VLCPUFA). In this study, the contribution of species with each type of fatty acids to the total ceramides (Cer) and sphingomyelins (SM) was investigated in rat and mouse testis and in rat spermatozoa. The major VLCPUFA in both lipids of testis were N- and 2-OH versions of 28:4n−6, 30:5n−6 and 32:5n−6 in the rat, and predominantly of 30:5n−6 in the mouse. Absent altogether from rat pre-puberal testes, SM and Cer with N-VLCPUFA appeared 10 days earlier than those with 2-OH VLCPUFA in postnatal development, in association with germ cell differentiation. Conversely, in adult fertile rats that were gradually deprived of germ cells in vivo after treatment with doxorubicin, SM and Cer with N-VLCPUFA decreased earlier than their 2-OH counterparts, and neither was present in aspermatogenic testes. In rat epididymal spermatozoa, the content of Cer prevailed over that of SM and 2-OH VLCPUFA prevailed over N-VLCPUFA in both lipids. In mature gametes, the acrosomal reaction resulted in an almost complete hydrolysis of the species of SM that contain both types of VLCPUFA to produce the corresponding Cer. Ceramides are biosynthetic precursors of SM in the testis, but themselves final products in spermatozoa. VLCPUFA-rich SM and Cer are thus produced in germ cells with the teleological objective of fulfilling their ultimate physiological role in spermatozoa that are apt and ready to fertilize an oocyte.     

A colorimetric assay for measuring phospholipase A2 degradation of phosphatidylcholine at physiological pH

Phospholipase A(2) is an important enzyme in various pathologies. Although fluorescent substrate assays for it have been recently developed, there is a need for an assay with inexpensive commercially available substrates, useful when samples interfered with fluorescent assays, that is nonisotopic, continuous, conducted at physiological pH, and in a 96 well format. A reaction using bromothymol blue was developed that meets all these requirements.     

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: A synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20° to 36 °C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 °C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

Oxidized phosphatidylcholine stimulates activity of secretory phospholipase A2 group IIA and abolishes sphingomyelin-induced inhibition of the enzyme

We have shown recently that oxidized but not native lipoproteins stimulate the activity of secretory phospholipase A2 group IIA (sPLA₂(IIA)). Since oxidized lipoproteins potentially contain considerable amounts of oxidized phosphatidylcholine, we examined the effect of oxidized palmitoyl arachidonyl phosphatidylcholine (oxPC) and the competitive effects of oxPC and sphingomyelin (SM) on sPLA₂(IIA) activity.OxPC either added to the assay medium as separated liposomes or incorporated in varied amounts into LDL progressively enhanced the activity of purified human sPLA₂(IIA) and abolished the inhibitory effect of LDL-incorporated SM on the enzyme activity. OxPC completely abolished the inhibitory effect of SM at the oxPC/SM concentration ratio 1/2. On the other hand, SM suppressed the activating effect of oxPC in a dose-dependent manner, abolishing it almost completely at a concentration 8 times as high as that of oxPC.Thus, changes in the oxPC/SM concentration ratio in LDL may affect the regulatory mechanisms of sPLA₂(IIA) activity in human blood, inducing stimulation or inhibition of the enzyme. Influence on regulation of sPLA₂(IIA) activity can be useful in the development of new therapeutic approaches to the treatment of cardiovascular diseases.     

Location and dynamics of acyl chain NBD-labeled phosphatidylcholine (NBD-PC) in DPPC bilayers. A molecular dynamics and time-resolved fluorescence anisotropy study

100-ns molecular dynamics simulations of fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, both pure and containing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl-chain labeled fluorescent analogs (C6-NBD-PC and C12-NBD-PC), are described. These molecules are widely used as probes for lipid structure and dynamics. The results obtained here for pure DPPC agree with both experimental and theoretical published works. We verified that the NBD fluorophore of both derivatives loops to a transverse location closer to the interface than to the center of the bilayer. Whereas this was observed previously in experimental literature works, conflicting transverse locations were proposed for the NBD group. According to our results, the maximum of the transverse distribution of NBD is located around the glycerol backbone/carbonyl region, and the nitro group is the most external part of the fluorophore. Hydrogen bonds from the NH group of NBD (mostly to glycerol backbone lipid O atoms) and to the nitro O atoms of NBD (from water OH groups) are continuously observed. Rotation of NBD occurs with ∼ 2.5-5 ns average correlation time for these probes, but very fast, unresolved reorientation motions occur in < 20 ps, in agreement with time-resolved fluorescence anisotropy measurements. Finally, within the uncertainty of the analysis, both probes show lateral diffusion dynamics identical to DPPC.     

Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2

Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.     

Human plasma lecithin-cholesterol acyltransferase. Inhibition of the phospholipase A2-like activity by sn-2-difluoroketone phosphatidylcholine analogues

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.D., and Dolphin, P.J. (1987) Biochim. Biophys. Acta 918, 175-188). This proposal is evaluated here by use of nonhydrolyzable sn-2-difluoroketone phosphatidylcholine analogues, known to inhibit calcium-dependent phospholipase A2. These compounds inhibited the calcium-independent phospholipase A2 activity of LCAT in a time and concentration dependent manner. The most potent analogues had a 100-fold higher affinity for the enzyme than the substrate, lecithin, when present within lecithin/apoA-I proteoliposomes. The inhibition was dependent upon the presence of a difluoromethylene group alpha to the sn-2-carbonyl carbon of the analogues. The inhibition is attributed to the formation of a tetrahedral adduct between the catalytic serine residue of LCAT and the sn-2-carbonyl carbon atom of the analogues which is stabilized by the electronegative fluorine atoms present upon the carbon atom alpha to the carbonyl carbon. This adduct mimics that proposed by us to occur during lecithin cleavage by LCAT, and the data substantiate the existence of this transition state adduct prior to the release of lysolecithin and formation of a fatty acylserine oxyester of the enzyme.     

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers

The interaction of the cationic tridecapeptide -melanocyte stimulating hormone (-MSH) and the biologically more active analog [Nle⁴, DPhe⁷]--MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5^th and 12^th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the -deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.     

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A₂ from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A₂ from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor

Summary The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the -amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.     

A subnanogram assay for phospholipase activity based on a long-chain radioiodinatable phosphatidylcholine

Here, we introduce a radioiodinatable long-chain phosphatidylcholine (BHC12PC) which serves as the base for a very sensitive phospholipase assay. This compound has a 4-hydroxyphenyl group attached at the end of the fatty acyl chain located in position sn-2. This feature enables this phospholipid to be radioiodinated. BHC12PC was tested as a substrate of Naja naja naja PLA(2) and Bacillus cereus PLC in a mixed micellar system with Triton X-100. The detection limit for the assays was 0.25ng of PLA(2) and 0.05ng of PLC, thus becoming one of the most sensitive methods described so far. A low specific radioactivity (500microCi/mmol) suffices to achieve this level of sensitivity. In both cases, the behavior of BHC12PC was indistinguishable from that shown by phospholipids with n-acyl chains of similar length. The choice of spacer prevents any unfavorable interaction of the bulky 4-hydroxyphenyl group at the active site of the enzymes. The progress of the reaction as monitored by thin-layer chromatography is compared side by side with an alternative method based on the selective adsorption of BHC12PC to silica gel, which renders identical results in a simpler fashion. An additional advantage of BHC12PC is that the cost per Ci of the radioiodinated derivative is significantly lower than that of other labeled phospholipids ((3)H, (14)C, or (32)P).