Search PubMed⌕ Search

Biomedical subjects

F Schroeder

Publications and source records attributed to F Schroeder.

At least 127 records · Page 7Linked to original sources
PubMed1990

Role of polyunsaturated fatty acids and lipid peroxidation in LM fibroblast plasma membrane transbilayer structure.

The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospholipids by culture medium supplementation, decreased the limiting anisotrophy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H2O2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbilayer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20-28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72-76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.

Animals↗
PubMed1990

Role of acidic phospholipids in intermembrane sterol transfer.

A liposomal membrane model system was used to examine the effect of acidic phospholipids on spontaneous intermembrane cholesterol transfer. The spontaneous exchange of sterol between small unilamellar vesicles (SUV) containing 35 mol% sterol was monitored with a recently developed assay (Nemecz, G., Fontaine, R.N. and Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511-541), not requiring separation of donor and acceptor membrane vesicles. Acidic phospholipids (2.5-30 mol%) increased the initial rate of spontaneous exchange of sterol by 5-89%, depending on the specific phospholipid. The stimulation of spontaneous sterol transfer by acidic phospholipids was suppressed by high ionic strength, CaCl2 and low pH. The results suggest that negatively charged phospholipids may fluidize sterol-poor domains in SUV membranes and thereby play an important role in the mechanism whereby sterols desorb from membranes into the aqueous medium.

Biological Transport↗
PubMed1990

The interaction of bile salt micelles with the dansyltyrosine derivatives of porcine colipase.

The interaction of bile salt micelles with the tyrosines of pancreatic colipase was assessed by steady-state and time-resolved fluorescence techniques. Dansyltyrosine fluorescence showed that Tyr-55 was located in the proposed interface recognition site. In support of this claim was a 70 nm blue shift and 4.3-fold quantum yield increase in emission spectrum due to taurodeoxycholate (TDOC) micelle-complex formation. Complex formation also caused a shift in the center of the major lifetime distribution from 11.7 to 15.1 ns, and more than doubled the polarization and anisotropy decay parameters. These data supported an earlier model of colipase-micelle binding that suggested that Tyr-55 was inserted into the interior of the TDOC micelle upon binding (J.C. McIntyre, P. Hundley and W.D. Behnke, Biochem. J. 245 (1987) 821). Identical experiments on a DNS-Tyr-59 derivative of colipase showed that Tyr-59 did not specifically interact with micelles. Moreover, acrylamide quenching data suggest an alteration in the protein environment surrounding DNS-Tyr-59 such that during complex formation, the efficiency of quenching of DNS-Tyr-59 increases.

Acrylamides↗
PubMed1989

Interaction of sphingomyelins and phosphatidylcholines with fluorescent dehydroergosterol.

The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)

Ergosterol↗
PubMed1989

Membrane properties of dolichol in different age groups of mice.

Dolichol content and the effects of dolichol in vitro on synaptic plasma membrane (SPM) structure of different age groups of C57BL/6NNIA mice were examined. SPM of aged mice had higher amounts of dolichol as compared to younger animals. Age differences also were observed in dolichol isoprenologue distribution. In older animals, dolichol isoprenologue chain length was shifted towards shorter chain length values. The structure of SPM was determined by differential polarized phase fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). The SPM of old mice were more rigid, as indicated by higher limiting anisotropy, than those of young mice. Dolichol administered in vitro to SPM fluidized the membranes. However, dolichol administered in vitro fluidized SPM of younger mice significantly more than SPM of aged mice. The functional role of increased dolichol content in aged individuals is not known. Dolichol may act to regulate membrane fluidity of aged animals.

Aging↗
PubMed1989

Acute and chronic effects of ethanol on transbilayer membrane domains.

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.

Alcohol Drinking↗
PubMed1988

Transbilayer effects of ethanol on fluidity of brain membrane leaflets.

Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.

Animals↗
PubMed1988

Time-resolved fluorescence investigation of membrane cholesterol heterogeneity and exchange.

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was investigated as a cholesterol analogue to examine sterol domains in and spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Fluorescence lifetime, acrylamide quenching analyses, and intermembrane exchange kinetics were consistent with the presence of at least two sterol domains in POPC. Fluorescence lifetime was determined by phase and modulation fluorescence spectroscopy and analyzed by nonlinear least-squares as well as continuous distributional analyses. Both methods demonstrated that pure dehydroergosterol in POPC SUV had two lifetime components (C) and fractional intensities (F) near C1 = 0.851 ns (F1 0.96) and C2 = 2.668 ns (F2 0.004). In contrast to component C1, the center of lifetime distribution, fractional intensity, and peak width of dehydroergosterol lifetime component C2 was dependent on the polarity of the medium and vesicle curvature. The sterol domain corresponding to dehydroergosterol component C2 was preferentially quenched by acrylamide. Acrylamide quenching of dehydroergosterol fluorescence demonstrated that the two lifetime components of dehydroergosterol were not due to transbilayer sterol domains with different lifetimes. In a spontaneous exchange assay not requiring separation of donor and acceptor SUV, the lifetime component C2, but not C1, shifted to a shorter lifetime with altered distributional width. The kinetics of these lifetime and distributional width changes best fitted a two-exponential function, with a fast exchange rate constant K1 = 0.0325 min-1, t1/2 = 21.3 min, and a slow rate constant k2 = 0.00275 min-1, t1/2 = 261 min. The fast exchanging pool correlates with the longer lifetime component C2. These kinetics were confirmed both by dehydroergosterol exchange measured with fluorescence intensity and by [3H]cholesterol exchange. In summary, lifetime, distributional width, acrylamide quenching, and classical exchange assay data are consistent with the presence of at least two pools of sterol in POPC SUV.

Cholesterol↗
PubMed1988

A fluorescence and radiolabel study of sterol exchange between membranes.

The fluorescent sterols delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) and delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) as well as [1,2-3H]cholesterol were utilized as cholesterol analogues to examine spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Exchange of fluorescent sterols was monitored at 24 degrees C by release from self-quenching of polarization from the time of mixing without separation of donor and acceptor vesicles. The polarization curve for 35 mol% sterol in POPC best fitted a two-exponential function, with a fast-exchange rate constant k1 = 0.0217 min-1, 1t1/2 = 32 min, size pool 1 = 12%, and a slow rate constant k2 = 2.91.10(-3) min-1, 2t1/2 = 238 min, size pool 2 = 88%. In addition to the above two exchangeable pools of sterol, the data were consistent with the presence of a slowly or nonexchangeable pool, 42% of total sterol, that was highly dependent on sterol content. These results were confirmed by simultaneous monitoring of [1,2-3H]cholesterol radioactivity and dehydroergosterol fluorescence intensity after separation of donor and acceptor vesicles by ion-exchange column chromatography. Thus, dehydroergosterol or cholestatrienol exchange as measured by fluorescence parameters (polarization and/or intensity) provides two new methods to follow cholesterol spontaneous exchange. These methods allow resolution and quantitation of a shorter exchange t1/2 near 30 min previously not reported. Thus, the cholesterol desorption rate from membranes may be faster than previously believed. In addition, the presence of a slowly non-exchangeable pool was confirmed.

Cholesterol↗
PubMed1988

Local and metastatic tumor growth and membrane properties of LM fibroblasts in athymic (nude) mice.

LM fibroblasts grown in a chemically-defined, serum-free medium readily incorporated choline or one of three analogues of choline, namely N,N-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine into membrane phospholipids. The effect of these phospholipid manipulations in vitro on tumor growth and metastasis was examined in nude mice. Serum and choline-fed cells most frequently metastasized (74% and 68%, respectively), while frequency of lung metastasis was 46%, 42% and 17% in mice injected with cells fed with dimethylethanolamine, monomethylethanolamine, and ethanolamine, respectively. Metastases from cells cultured with serum, choline or dimethylethanolamine, but not from monomethylethanolamine or ethanolamine, were extensive and highly invasive. The specific activity of the (Na+ + K+)-ATPase but not of 5'-nucleotidase was significantly decreased in local tumor plasma membranes from choline analogue-fed cells as compared to tumor plasma membranes from choline-fed cells. When compared to the choline-fed tumor cells, the specific activities of three mitochondrial enzymes, namely NADH dependent, rotenone insensitive NADH-dependent, and rotenone sensitive NADH-dependent cytochrome-c reductase, were significantly increased in the choline analogue-supplemented cells. The arachidonic acid content of phosphatidylcholine in plasma membranes, microsomes, and mitochondria was significantly decreased in tumor membranes from choline analogue-fed cells as compared to tumor membranes from choline-fed cells. As compared to local tumor plasma membranes, the lung metastasis plasma membranes had elevated (Na+ + K+)-ATPase specific activity, phospholipid oleic and arachidonic acid content, and fluidity. In contrast, the 5'-nucleotidase specific activity, the content of cholesterol, phospholipid, and phosphatidylethanolamine were decreased in lung metastasis plasma membranes. In summary, membrane alterations of LM tumor cells in vitro (1) were not completely reversed in vivo, and (2) affected metastatic ability.

5'-Nucleotidase↗
PubMed1988

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane.

Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to min, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate- or linolenate-supplemented medium, 71.8% and 75.5% of the plasma membrane sterol is exofacial, respectively.

Animals↗
PubMed1988

Membrane effects of ethanol: bulk lipid versus lipid domains.

It has been well-established that ethanol fluidizes the bulk lipid of membranes and that this effect may alter cell function and be involved in ethanol sensitivity and tolerance. This hypothesis has been supported in several studies, however, there is also a considerable amount of data that do not support such an explanation, e.g., direct effect of ethanol on proteins, other membrane acting drugs, temperature effects, effects of ethanol on aged membranes and inconsistent effects of chronic ethanol consumption on lipid content. This review examined the bulk membrane fluidization hypothesis in light of those data and proposed a modification of the bulk membrane hypothesis that is based on recent data that show that ethanol and other alcohols have a specific effect on the structural properties of different membrane domains. This specific effect of ethanol is discussed within the context of how changes in fluidity of domains may alter membrane function.

Aging↗
PubMed1988

Fluorescence properties of cholestatrienol in phosphatidylcholine bilayer vesicles.

The fluorescent sterol delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) was incoporated into 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. Previously another fluorescent sterol, dehydroergosterol (F. Schroeder, Y. Barenholz, E. Gratton and T.E. Thompson. Biochemistry 26 (1987) 2441), was used for this purpose. However, there is some concern that dehydroergosterol may not be the best analogue for cholesterol. Fluorescence properties of cholestatrienol in POPC SUV were highly sensitive to cholestatrienol purity. The fluorescence decay of cholestatrienol in the POPC SUV was analyzed by assuming either that the decay is comprised of a discrete sum of exponential components or that the decay is made up of one or more component's distribution of lifetimes. The decay for cholestatrienol in POPC SUV analyzed using distributions had a lower chi 2 value and was described by a two-component Lorentzian function with centers near 0.86 and 3.24 ns, and fractional intensities of 0.96 and 0.04, respectively. Both distributions were quite narrow, i.e., 0.05 ns full-width at half-maximum peak height. It is proposed that the two lifetime distributions are generated by separate continua of environments for the cholestatrienol molecule described by different dielectric constants. In the range 0-6 mol% cholestatrienol, the cholestatrienol underwent a concentration-dependent relaxation. This process was characterized by red-shifted absorption and maxima and altered ratios of absorption and fluorescence excitation maxima. Fluorescence quantum yield, lifetime, steady-state anisotropy, limiting anisotropy and rotational rate remained constant. In contrast, in POPC vesicles containing between 6 and 33 mol% cholestatrienol, the fluorescent cholestatrienol partially segregated, resulting in quenching. Thus, below 6 mol% cholestatrienol, the cholestatrienol appeared to behave in part as monomers exposed to some degree to the aqueous solvent in a sterol-poor domain within POPC bilayers. Since the lifetime did not decrease above 6 mol% cholestatrienol, the fluorescence at high mol% values of cholestatrienol was due to cholestatrienol in the sterol-poor domain. The fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy of cholestatrienol in the sterol-poor domain decreased to limiting, nonzero values while the rotational rate increased to a limiting value. Thus, the sterol-poor domain became more disordered when it coexisted with the sterol-rich domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholestenes↗
PubMed1987

The influence of dolichols on fluidity of mouse synaptic plasma membranes.

Dolichols are isoprenologues which constitute an important component of biological membranes. However, an understanding of the effects of dolichols on the organization and dynamics of biological membranes has not been forthcoming. The experiments reported here are aimed at understanding the effects of dolichols on the physical properties of mouse brain synaptic plasma membranes. The effect of dolichols incorporated into mouse brain synaptic plasma membranes on fluorescent and electron spin resonance probes sensing the hydrophobic core differed from that of probes reporting closer to the surface of membrane bilayers. Dolichols significantly (P less than 0.01) lowered the polarization, limiting anisotropy, and order parameter of diphenylhexatriene in synaptic plasma membranes and liposomes extracted from synaptic plasma membranes, without changing the rotational relaxation time. Similarly, dolichol increased the fluidity reported by 16-doxylstearic acid in synaptic plasma membranes or liposomes extracted from synaptic plasma membranes. In contrast, dolichols exerted no effect on those properties for trans-parinaric acid or 5-doxylstearic acid in synaptic plasma membranes or liposomes derived therefrom. Dolichols can dramatically alter the structure and dynamics of lipid motion in synaptic plasma membranes and these effects are dependent on the location of the probe in the membrane.

Animals↗
PubMed1987

Charged anesthetics selectively alter plasma membrane order.

Although indirect evidence supporting differential lipid fluidity in the two monolayers of plasma membranes has accumulated, unambiguous demonstration of this difference has been difficult to obtain. In the present study, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), selective quenching of fluorescence by trinitrophenyl groups, and differential polarized phase fluorescence techniques were used to directly examine the static (order) and dynamic (rotational rate) components of lipid motion in the exofacial and cytofacial leaflets of LM fibroblast plasma membranes. The limiting anisotropy (0.137), the order parameter (0.590), and the rotational relaxation time (1.20 ns) of DPH in the plasma membranes (inner plus outer leaflet) indicated rapid but restricted probe motion in the lipid environment. However, the statics and dynamics of DPH motion in the individual monolayers were significantly (p less than 0.025) different. The limiting anisotropy, order parameter, and rotational relaxation time of DPH in the cytofacial monolayer were 0.036, 0.08, and 0.16 ns, respectively, greater than calculated for the exofacial monolayer of the LM plasma membrane. At appropriate concentrations, phenobarbital and, to a lesser degree, pentobarbital preferentially reduced the limiting anisotropy of DPH calculated for the exofacial leaflet while prilocaine reduced the limiting anisotropy of DPH in the cytofacial leaflet of LM fibroblast plasma membranes. In contrast, the putative cytofacial anesthetic procaine failed to show any preference for either leaflet. Arrhenius plots of DPH fluorescence in LM plasma membranes showed a prominent characteristic break point near 30-32 degrees C. Phenobarbital, pentobarbital, and procaine did not affect this break point while prilocaine selectively abolished it. The break point was therefore assigned to the inner monolayer of the LM plasma membrane.

Anesthetics↗
PubMed1987

A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles.

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was incorporated into 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. In the range 0-5 mol % fluorescent sterol, dehydroergosterol underwent a concentration-dependent relaxation characterized by red-shifted wavelengths of maximum absorption as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338 nm/324 nm. Fluorescence intensity per mole of dehydroergosterol increased up to 5 mol % in POPC vesicles. In contrast, quantum yield, steady-state anisotropy, limiting anisotropy, lifetime, and rotational rate remained relatively constant in this concentration range. Similarly, addition of increasing cholesterol in the range 0-5 mol % in the presence of 3 mol % dehydroergosterol also increased the fluorescence intensity per mole of dehydroergosterol, red-shifted wavelengths of maximum absorption, and altered ratios of absorbance maxima. In POPC vesicles containing between 5 and 33 mol % dehydroergosterol, the fluorescent dehydroergosterol interacted to self-quench, thereby decreasing the fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy and increasing the rotational rate (decreased rotational relaxation time) of the fluorescent sterol. The fluorescence lifetime of dehydroergosterol remained unchanged. The results were in accord with the interpretation that below 5 mol% sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol↗
PubMed1987

Composition and properties of very low density lipoproteins secreted by the perfused rat liver and subfractionated by affinity chromatography.

Very low density lipoproteins (VLDL) were isolated from the perfusate of rat livers infused with a complex of oleic acid bound to bovine serum albumin. Very low density lipoprotein (VLDL) secretion, bile flow, histopathology, and transmission electron microscopy indicated that secretory functions but not morphologic integrity of the livers were maintained during the procedure. Plasma VLDL and liver perfusate VLDL did not have similar size distribution. VLDL isolated from recycling perfusate and single pass perfusate were also subfractionated with concanavalin A-Sepharose 4B affinity chromatography. Three subfractions were eluted sequentially from the perfusate VLDL: a non-adherent fraction A and two adherent fractions B and C. The size of these VLDL, determined after negative staining and examination by transmission electron microscopy, was significantly decreased by affinity chromatography. VLDL in fractions A, B and C were spherical and had diameters of 935 +/- 17, 881 +/- 34 and 415 +/- 30 A respectively. Fraction A, which did not adhere to the column, contained 65% of the lipid applied to the column. The carbohydrate composition of fraction A VLDL was 11.2 +/- 0.6% fucose, 14.7 +/- 1.2% galactose, 43.7 +/- 2.3% N-acetylglucosamine, and 30.5 +/- 1.9% sialic acid. Sugars such as glucose and mannose, which bind to concanavalin A, were not detected. In contrast, VLDL fractions B and C, which adhered to the column, contained both glucose (17.7 and 2.5%) and mannose (5.8 and 8.3%) as well as the other sugars present in VLDL fraction A. Sodium dodecyl sulfate gradient gel electrophoresis revealed that the affinity column procedure clearly altered the apolipoprotein patterns of the applied VLDL, thereby producing abnormal fractions B and C. Fractions B and C also differed from unfractionated VLDL and fraction A VLDL in lipid composition, in surface/interior core lipid ratio, and in fatty acid composition of the interior core lipids, primarily triacylglycerols. The steady-state anisotropy, the limiting anisotropy and the lipid order parameter of fluorescence probe molecules 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid incorporated into the VLDL were of the following order: fraction B greater than fraction A greater than fraction C. These results are consistent with the interpretation that concanavalin A-Sepharose 4B affinity chromatography may artificially produce a series of VLDL subfractions whose composition and structural properties do not resemble those of native VLDL.

Animals↗