Lipid analysis of bronchoalveolar lavage fluid (BAL) by MALDI-TOF mass spectrometry and 31P NMR spectroscopy

Despite the high clinical relevance, only the cellular moiety of bronchoalveolar lavage (BAL) has been intensively investigated and is used for diagnosis purposes. On the other hand, the cell-free fluid is, by far, less characterized. Although this fluid represents a relatively simple mixture of only a few different phospholipids (mainly phosphatidylcholine, phosphatidylglycerol and cholesterol), methods for the routine analysis of these fluids are still lacking. In the present investigation we have applied, for the first time, MALDI-TOF mass spectrometry, as well as 31P NMR spectroscopy to the analysis of organic extracts of bronchoalveolar lavage fluids. BAL from different mammals (rat, minipig, rabbit and man) were investigated and, for means of comparison, organic extracts of lung tissue were also examined. Both applied methods provide fast and reliable information on the lipid composition of the bronchoalveolar lavage. However, despite of its comparably low sensitivity, 31P NMR spectroscopy detects all phospholipid species in a single experiment and with the same sensitivity, whereas MALDI-TOF fails in the detection of phosphatidylethanolamine in the presence of higher quantities of phosphatidylcholine. In contrast, MALDI-TOF mass spectrometry is more suitable for the detection of cholesterol and the determination of the fatty acid composition of the individual phospholipids, especially lysolipids. It will be shown that all BALs exhibit significant, species-dependent differences that mainly concern the content of phosphatidylglycerol and lyso-phosphatidylcholine. It is concluded that both methods are suitable tools in lipid research due to the (in comparison to alternative methods) simplicity of performance.     

Lipid analysis of human HDL and LDL by MALDI-TOF mass spectrometry and (31)P-NMR

The analysis of HDL and LDL is important for the further understanding of atherosclerosis because changes of the protein and lipid moieties occur under pathological conditions. Because destruction of lipids leads to the formation of well-defined products such as lysophospholipids or chlorohydrins, methods that allow their fast and reliable determination would be useful. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for the analysis of the lipid composition of human lipoproteins. These data were compared with high resolution (31)P-NMR spectroscopy. Differences between LDL and HDL in sphingomyelin and phosphatidylcholine content could be monitored by NMR and mass spectrometry, and differences with respect to the extraction efficiency were found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A(2) resulted in marked changes (formation of chlorohydrines and lysolipids). Lysophosphatidylcholines were detectable by both methods, whereas MALDI-TOF MS failed to detect chlorohydrines of phospholipids.We conclude that MALDI-TOF MS provides rapidly a reliable lipid profile of lipoproteins. However, a previous lipid separation must be performed to detect lipid oxidation products. NMR can be directly applied, but suffers from lower sensitivity, and provides only limited information on fatty acid composition.     

Separation of phosphatidylinositols and other phospholipids by two-step one-dimensional thin-layer chromatography

A quick and efficient thin-layer chromatographic procedure is described for the separation of phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol, phosphatidylcholine, phosphatidic acid, and phosphatidylethanolamine. The method involves development of the phospholipids successively in two different solvent systems but in the same direction. The method is simple, reproducible, and gives good resolution of the six different phospholipids tested. About 8-10 32P-labeled phospholipids isolated from rat hepatocytes were separated by this method; the six mentioned above were identified. Thus, the technique has potential application for both qualitative and quantitative analysis of phospholipid mixtures, such as the phosphoinositides, in cell or tissue extracts.     

Analysis of the lipid composition of bull spermatozoa by MALDI-TOF mass spectrometry--a cautionary note

In this study we demonstrated the combination of MALDI-TOF MS and TLC as a fast and powerful tool to investigate the phospholipid (PL) composition of organic extracts of bull spermatozoa. Since phosphatidylcholine (PC) is the dominant PL species, an adequate resolution of MALDI-TOF spectra for sphingomyelin (SM) or phosphatidylethanolamine (PE) was achieved only after previous PL separation by TLC. We found a poor diversity especially for PE and PC, mainly containing ether-linked fatty acids which were 1-palmityl-2-docosahexaenoyl-PL and the corresponding alkenyl-acyl compound (plasmalogen) 1-palmitenyl-2-docosahexaenoyl-PL. For PC, both lipids were quantified after phospholipase A2 digestion to represent 44.2 and 37.2%, respectively, of the total PC. In contrast, the diacyl-PC content of bull spermatozoa was comparatively low (18.6% of total PC). In the presence of trifluoroacetic acid (TFA), which is routinely added to the MALDI-TOF matrix to improve the signal to noise ratio, a high lysophospholipid (LPL) content was detected in the PL extracts of bull spermatozoa, whereas TLC did not reveal significant amounts of LPL. The TFA mediated hydrolysis of the acid-labile alkenyl-acyl PL to the corresponding LPL was shown to cause this discrepancy. This assumption was verified by analysing the PL composition by MALDI-TOF MS before and after (i) digestion of sperm cell lipids with phospholipase A2 and (ii) exposition of spermatozoa to HCl fumes. We conclude that the analysis of samples containing alkenyl-acyl-PL by MALDI-TOF has to be performed with great caution.     

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.     

Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for the detection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and a high phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as the minimum of analyte on the sample plate resulting in a signal-to-noise-ratio of S/N=5. Using 2,5-dihydroxybenzoic acid (DHB) as matrix the detection limit for phosphatidylinositol (PI) is seven times higher than for phosphatidylcholine (PC) and further increases with increasing phosphorylation or in mixtures with other well-detectable phospholipids. For phosphatidylinositol-tris-phosphate (PIP₃) in a mixture with PC, the limit is about 20 times higher than for PI. The consequences for the experimental conditions are discussed. It is advisable to pre-separate PPI from biological lipid mixtures prior to the application of MALDI-TOF MS.     

Separation and quantitation of phospholipids and lysophospholipids by high-performance liquid chromatography

We describe a comprehensive approach to the separation, quantitation, and characterization of phospholipids and lysophospholipids present in complex biological samples. The central feature is a normal-phase HPLC separation of individual phospholipid and lysophospholipid classes. In this single chromatographic step, phospholipids and lysophospholipids are separated and recovered for quantitation by organic phosphate assay and characterization by acyl-group composition. Recovery of phospholipids and lysophospholipids from HPLC averages 80-90%. Isolated phospholipid and lysophospholipid fractions are available for separation of individual molecular species by second-dimension reverse-phase HPLC and characterization of individual molecular species by mass spectrometry.     

Characterization of human lysophospholipid acyltransferase 3

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K_0.5 (5 μM), a monounsaturated acyl-CoA having the highest apparent V_max (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.     

Resistance to ethanol disordering of membranes from ethanol-fed rats is conferred by all phospholipid classes.

Phospholipids extracted from liver microsomes and mitochondria of ethanol-fed rats retained the resistance to membrane disordered by ethanol which is observed in the intact isolated membranes. The lipid extracts were separated into the major phospholipid classes (phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol from microsomes and phosphatidylcholine, phosphatidylethanolamine and cardiolipin from mitochondria) by preparative TLC. The extent of membrane disordering by ethanol of phospholipid vesicles composed of a mixture of phospholipids from ethanol-fed rats and controls was determined from the reduction of the order parameter of the spin-probe 12-doxyl-stearate. In contrast to previous reports, we found that all phospholipid classes from ethanol-fed rats confer resistance to disordering by ethanol. To a first approximation the extent of resistance was proportional to the fraction of lipids from ethanol-fed rats, regardless of the phospholipid head-group. Subtle differences between phospholipid classes may exist but were too small to measure accurately. Except for phosphatidylethanol, incorporation of anionic phospholipids did not have a significant effect on the sensitivity of phospholipid vesicles to the disordering effect of ethanol. Vesicles prepared from mixtures of various dioleoyl phospholipids and natural phospholipids did not indicate a clear effect of fatty acid saturation on the sensitivity to disordering by ethanol. Although the precise molecular changes that occur in phospholipids from ethanol-fed rats have not been fully characterized it appears that subtle changes in all phospholipid classes contribute to the resistance to ethanol disordering of these membranes.     

Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR

The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.     

Negatively charged phospholipids and their position in the cholesterol affinity sequence

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyelin with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin.From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin ? phosphatidylserine, phosphatidylglycerol > phosphatidylcholine ? phosphatidylethanolamine.     

Identification of oxidized phospholipids in bronchoalveolar lavage exposed to low ozone levels using multivariate analysis

Chemical reactions with unsaturated phospholipids in the respiratory tract lining fluid have been identified as one of the first important steps in the mechanisms mediating environmental ozone toxicity. As a consequence of these reactions, complex mixtures of oxidized lipids are generated in the presence of mixtures of non-oxidized naturally occurring phospholipid molecular species, which challenge methods of analysis. Untargeted mass spectrometry and statistical methods were employed to approach these complex spectra. Human bronchoalveolar lavage (BAL) was exposed to low levels of ozone, and samples with and without derivatization of aldehydes were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry. Data processing was carried out using principal component analysis (PCA). Resulting PCA scores plots indicated an ozone dose-dependent increase, with apparent separation between BAL samples exposed to 60 ppb ozone and non-exposed BAL samples as well as a clear separation between ozonized samples before and after derivatization. Corresponding loadings plots revealed that more than 30 phosphatidylcholine (PC) species decreased due to ozonation. A total of 13 PC and 6 phosphatidylglycerol oxidation products were identified, with the majority being structurally characterized as chain-shortened aldehyde products. This method exemplifies an approach for comprehensive detection of low-abundance, yet important, components in complex lipid samples.     

Ammonia desorption chemical ionization mass spectrometry of phospholipids

Ammonia desorption chemical ionization mass spectra (NH₃-DCIMS) of phosphatidyl-sulfocholines, a mixture of a homologous series of phosphatidylsulfocholines (PSC) and a mixture of a PSC phosphatidylcholine (PC), were measured by a flash heating method involving introduction of 1 μg of the samples on a tungsten wire, quickly heated, into the ion source of a Finnigan 4000/INCOS quadrupole instrument. It was possible to observe mass spectra during several seconds.The first spectra recorded of the PSCs gave essentially only the quasi-molecular [M + 18]⁺ peaks and the ‘A’ (see text) peaks. Spectra of a mixture of three homologous PSCs clearly showed three pairs of the above peaks. In contrast spectra of the PCs gave principally the [M + 1]⁺ and the ‘A’ peaks after 2s so that one can distinguish the diagnostic peaks in a mixture of a PC and a PSC.These results show that ammonia desorption chemical ionization by fast heating is a suitable technique for the charaterization of some head groups of phospholipids as well as a promising method for the analysis of phospholipid mixtures.     

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound. Dedicated to Prof. Dr. Klaus Arnold on the occasion of his 65th birthday.     

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.     

CsCl as an auxiliary reagent for the analysis of phosphatidylcholine mixtures by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is mainly used for protein and peptide analysis. However, there is growing evidence that also phospholipids like phosphatidylcholines (PC) can be easily analyzed by MALDI-TOF MS. In MALDI-TOF methodology, the sample is cationized by the addition of inorganic ions. This process is strongly dependent on the corresponding ion concentration. In biological samples various cations are present (mainly H+, Na+ and K+) and, therefore, a mixture of different adducts is formed. Since phospholipids exhibit a wide distribution of different fatty acid residues a considerable peak overlap may occur. This is a major problem since the peak assignment in a mixture will be often unclear. In this paper we demonstrate that this problem can be easily overcome by mixing the analyte with caesium chloride (CsCl). This yields naturally non-occurring Cs+ adducts that are apparent due to the large shift of the molecular mass. The proposed method facilitates the clear assignment of most peaks. Besides that, we will show that CsCl can also be used for the determination of the relative fatty acid composition of a given PC sample. For this purpose naturally occurring mixtures of PCs as well as organic extracts of human lipoproteins-that are mainly composed of PC and sphingomyeline-are used.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Investigation of phospholipids of the pulmonary extracellular lining by electron paramagnetic resonance. The effects of phosphatidylglycerol and unsaturated phosphatidylcholines on the fluidity of dipalmitoyl phosphatidylcholine.

The membranous structures of the pulmonary extracellular lining were removed from the lungs of rabbits by pulmonary lavage and isolated by differential centrifugation. This membranous fraction contained 93% of the total extracellular phospholipids present in lavage effluents and consisted of membranous vesicles, membrane fragments, tubular myelin and secreted lamellar bodies. The fraction was rich in phosphatidylcholine (79.4%) containing 85.2% palmitic acid in the 1-position and 57.4% palmitic acid in the 2-position. Phosphatidylglycerol was the next most abundant phospholipid, accounting for 9.4% of the total. E.p.r. spectra, obtained by using 5-doxylmethylstearate as a probe, showed that the extracellular phospholipids of the pulmonary lining were organized into structures which were much more fluid than erythrocyte-ghost membranes. The fluidity of phosphatidylcholine isolated from the membranous fraction was similar to that of the fraction itself, indicating that the minor phospholipids had very little influence on the fluidity of the major phospholipid. At physiological temperature, the fluidity of dipalmitoyl phosphatidylcholine was relatively low, but could be markedly increased by the presence of 1-palmitoyl-2-oleoyl phosphatidylcholine or phosphatidylglycerol (10%). Protein present in the extracellular phospholipid fraction did not affect the fluidity of the fraction. These studies indicate that the unsaturated phosphatidylcholines could play a major role in determining the fluidity of the important surface-tension-lowering phospholipids such as dipalmitoyl phosphatidylcholine.     

Properties of membrane phospholipids and their fatty acyl compositions of secretory granules from rat parotid gland

A secretory granular fraction isolated from rat parotid glands was remarkably different from a microsomal fraction in its phospholipid composition. It had higher levels of lysophospholipids (8%) and phosphatidylethanolamine (31%), while there were lower levels of phosphatidylcholine (40%) and phosphatidylserine (2.1%) than the microsomal fraction. However, fatty acid compositions of individual phospholipid classes from the two subfractions were found to be nearly similar to each other. ESR analysis demonstrated that extracted phospholipids from the secretory granular fraction were more fluid than those from microsomes. The relevance of these observations to physiological function of secretory granules is discussed.     

Filamentous Fungi with High Cytosolic Phospholipid Transfer Activity in the Presence of Exogenous Phospholipid

The phospholipid transfer activity of cell extracts from 15 filamentous fungus strains grown on a medium containing phospholipids as the carbon source was measured by a fluorescence assay. This assay was based on the transfer of pyrene-labeled phosphatidylcholines forming the donor vesicles to acceptor vesicles composed of egg phosphatidylcholines. The highest phosphatidylcholine transfer activity was obtained with cell extracts from Aspergillus oryzae. The presence of exogenous phospholipids in the culture medium of A. oryzae was shown to increase markedly the activity of phospholipid transfer as well as the pool of exocellular proteins during the primary phase of growth. Modifications in the biochemical marker activities of cellular organelles were observed: succinate dehydrogenase, a mitochondrial marker; inosine diphosphatase, a Golgi system marker; and cytochrome c oxidoreductase, an endoplasmic reticulum marker, were increased 7.3-, 2-, and 22-fold, respectively, when A. oryzae was grown in the presence of phospholipids.