Analysis of phospholipids in bifidobacteria

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modem procedures for identifying Bifidobacterium species.     

Lipid oxidation in biological membranes. Electron transfer proteins as initiators of lipid autoxidation.

Biological lipid autoxidation has been studied in a model system composed of sonicated phospholipids as substrate and electron transfer proteins found in membranes as possible catalysts. Heme compounds, flavoproteins, and iron-sulfur proteins were examined for their ability to initiate lipid autoxidation. Among many heme compounds tested, the most active were hematin ?microperoxidase ? methemoglobin > cytochrome c. With fresh preparations of phospholipids, reaction rates (nanomoles of oxygen/minute nanomoles of heme) ranged from 5 (cytochrome c) to 350 (hematin). Only the oxidized heme compounds were active as catalysts. Reduced heme compounds, flavoproteins and riboflavin were inactive. In the presence of heme compounds, aged preparations of sonicated phospholipids were much more rapidly oxidized than fresh preparations. They also had a higher content of fatty acid hydroperoxides as judged from their characteristic diene absorption peak at 234 nm. This observation agrees with the postulated mechanism of lipid autoxidation by heme compounds, namely, homolytic scission of preformed fatty acid hydroperoxides. Iron-sulfur proteins were also active as initiators of lipid autoxidation when destabilized in the presence of an appropriate iron chelator (o-phenanthroline or 2,2′-bipyridine) or a chaotropic ion. Oxygen uptake rates (nanomoles of oxygen/minute × milligrams of protein) varied from about 200 for an iron-sulfur protein isolated from complex I to about 5500 for Clostridium pasteurianum ferredoxin. However, per nanomole of labile sulfide, the rates for all active iron-sulfur proteins were 4–7 nmol of oxygen/min × nmol of labile sulfide.Superoxide-generating systems did not initiate lipid autoxidation, nor did erythrocuprein inhibit the autoxidations induced by heme compounds or ferredoxin. However, lipid oxidations induced by two other iron-sulfur proteins were partially inhibited by erythrocuprein. It is concluded that in the above system Superoxide anion is neither an initiator nor an obligatory intermediate of lipid autoxidation.     

Isolation and Comparative Analysis of Glycolipid Fractions in Bifidobacteria

A comparative TLC analysis of lipid extracts from Bifidobacterium longum B 379 M, B. bifidum 791, and B. adolescentis 94 BIM has been performed. It is demonstrated that carbohydrate-containing lipid components were present in the bacteria, which differed in their chromatographic mobility (R _f ) from similar compounds isolated from actinomycetes Stomatococcus mucilaginosus PCM 2415^T, Nocardiopsis dassonvillei PCM 2492, Propionibacterium propionicum PCM 2431, Saccharopolyspora hirsuta PCM 2279 (= ATCC 27875^T), Rhodococcus equi PCM^T 559 (= ATCC 3969), and Gordonia bronchialis PCM 2167. Polar lipids of bifidobacteria exhibited the closest similarity to their counterparts from propionic acid bacteria. Preparative chromatography (silica gel column I; elution with chloroform, acetone, and methanol) of the lipid extract of B. adolescentis 94 BIM made it possible to isolate fractions containing nonpolar lipids, glycolipids, and phospholipids. Further purification of the glycolipid fraction (column II; eluant, methanol gradient in chloroform) produced preparations of glycolipids and phospholipids. The preparations were studied by two-dimensional TLC using solvent systems chloroform-methanol-H₂O MiLi Q (65 : 25 : 4, v/v/v) and n-butanol-acetic acid-H₂O MiLi Q (60 : 20 : 20, v/v/v) for directions I and II, respectively. Two major glycolipids were revealed (G₁ and G₂), in addition to compounds characteristic of the polar lipid group and minor glycolipids (g), the latter being present in considerably lesser amounts.     

Discontinuous thermotropic response of Tetrahymena membrane lipids correlated with specific lipid compositional changes

Steady-state fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in microsomal lipids from Tetrahymena pyriformis cells grown at 39 or 15°C revealed discrete slope discontinuities in plots of polarization vs. temperature. Two well-defined ‘break points’ were present in the 0–40°C temperature range examined and their precise location was dependent upon the growth temperature of the cells. By mixing phospholipids from cells grown at different temperatures, the break points at 17.5 and 32°C in 39°C-lipid multilayer preparations were shown to correlate with the breaks at 12 and 27°C, respectively, in similar preparations from 15°C-grown cells. The discrete break points were also present, but at slightly different characteristic temperatures, in a phosphatidylcholine fraction and a phosphatidylethanolamine plus 2-aminoethylphosphonolipid fraction purified from the phospholipids and in total microsomal lipids (phospholipids plus the sterol-like triterpenoid, tetrahymanol). However, catalytic hydrogenation of the phospholipid fatty acids or mixing the non-hydrogenated phospholipids with increasing proportions of synthetic dipalmitoyl phosphatidylcholine eliminated the break points. We interpret this discontinuous thermotropic response in microsomal lipids as signalling a lipid phase separation of importance in regulating physiological events.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Characterization of two cell-envelope fractions from chemotrophically grownRhodospirillum rubrum

Two cell-envelope fractions were isolated from chemotrophically grown cells ofRhodospirillum rubrum. On the basis of electron-microscopic investigations, chemical analysis, distribution of components involved in respiration, and polyacrylamide gel electrophoresis, the heavy fraction (ρ²⁰=1.246 g per cm³) was identified as cell-wall, and the light fraction (ρ²⁰=1.145 g per cm³) as cytoplasmic-membrane fragments. Electron micrographs showed cell-wall fragments as open structures while cytoplasmic-membrane preparations were composed of closed membrane vesicles. With respect to the main classes of chemical compounds, cell wall could be distinguished from cytoplasmic membranes by a rather low ratio of phospholipids per protein and a high ratio of carbohydrates per protein. The relative proportion of individual neutral sugars as well as phospholipids (except for lysophosphatidyl ethanolamine) revealed no significant differences between both envelope fractions. Fatty acid analysis demonstrated a higher proportion of saturated fatty acids in cell-wall than in cytoplasmic-membrane fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions showed distinct protein compositions. While in cell-wall preparations polypeptides of 43000 and 14000 daltons predominated, 56000- and 52000-dalton polypeptides were the main protein subunits of cytoplasmic membranes. Cross contaminations of both cell-envelope fractions were defined.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

Biochemical aspects of the visual process. XXXVII. Evidence for lateral aggregation of rhodopsin molecules in phospholipase C-treated bovine photoreceptor membranes

Photoreceptor membranes derived from isolated bovine rod outer segments, are subjected to treatment with phospholipase C (Bacillus cereus). This results in varying degrees of hydrolysis of the membrane phospholipids into diglycerides and water soluble phosphate esters without loss of rhodopsin. Electron microscopic observations of thin sections and freeze-fractured preparations indicate extrusion of diglycerides from the membranes and their coalescence to lipid droplets, beginning at 20% hydrolysis of phospholipids. After 90% hydrolysis of phospholipids membranous structures are still present. The rhodopsin is located in these structures, presumably in the form of two-dimensional lateral aggregates. This explains the cross-fracturing of the membranous structures, regularly observed upon freeze-fracturing of the phospholipase-treated photoreceptor membranes.     

Light-induced generation of a protonmotive force and Ca2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The light-driven primary proton pump bacteriorhodopsin has been incorporated in the cytoplasmic membrane of Streptococcus cremoris, in order to generate a protonmotive force across these membranes. This has been achieved by fusion of S. cremoris membrane vesicles with bacteriorhodopsin proteoliposomes. This fusion occurred when both preparations were mixed at low pH (less than 6.0), as shown by sucrose density gradient centrifugation and by dilution of fluorescent phospholipids incorporated into the bacteriorhodopsin proteoliposomes. Fusion was strongly enhanced by the presence of negatively charged phospholipids in the liposomal bilayer. When proteoliposomes were used that showed light-dependent proton uptake, the orientation of bacteriorhodopsin in the fused membranes was inside-out with respect to the in vivo orientation in Halobacterium halobium. Consequently, in the light a ΔΨ, interior positive and a ΔpH, interior acid were generated. This protonmotive force could drive calcium uptake in the fused membranes. The uptake increased hyperbolically with increasing light intensity and was abolished by bleaching of bacteriorhodopsin. Addition of the ionophore valinomycin stimulated calcium uptake and led to an increase of the ΔpH. Calcium uptake was strongly decreased in the dark and in the light in the presence of uncouplers, nigericin or both valinomycin and nigericin.     

Fatty acid composition and incorporation of arachidonic acid into phospholipids of hemocytes from the tobacco hornworm Manduca sexta

The fatty acid compositions were determined for total lipids, triacylglycerols, phospholipids and four phospholipid fractions, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/phosphatidylinositol (PS/PI) and cardiolipin (CA) obtained from hemocytes and cell-free serum from second day, fifth instar larvae of the tobacco hornworm Manduca sexta and the standard Manduca rearing medium. The hemocyte fatty acid profiles were considerably different from the profiles of the medium the insects were reared on and from the profiles of the cell-free serum. Hemocyte neutral lipids had lower proportions of polyunsaturated fatty acids than phospholipids. The fatty acid profiles of PC, PE, PS/PI and CA differ from each other and from the total lipid profiles, indicating selective fatty acid incorporation into hemocyte phospholipid species. Studies with radioactive arachidonic acid similarly indicated selective incorporation of polyunsaturated fatty acids into hemocyte lipids. Under our in vitro conditions, >40% of the total radioactivity was incorporated into hemocyte lipids. About 93% of the incorporated radioactivity was found in phospholipids. Within phospholipids. most of the radioactivity was associated with PC (46%), and less with PE (28%) and PS/PI (21%). Very little radioactivity was recovered in CA (0.9%).     

Insulin inhibits changes in the phospholipid profiles in sciatic nerves from streptozocin-induced diabetic rats: A phosphorus-31 magnetic resonance study

Sciatic nerve phospholipids obtained from insulin-treated streptozocin-induced diabetic, non-treated streptozocin-induced diabetic, and healthy, control male Sprague-Dawley rats after eighteen weeks of diabetes were studied by ³¹P NMR spectrometry. Eleven phospholipids resonances were identified as follows: Phosphatidic acid (Chemical shift, 0.30 ppm), dihydrosphingomyelin (0.13 ppm), ethanolamine plasmalogen (0.07 ppm), phosphatidylethanolamine (0.03 ppm), phosphatidylserine (−0.05 ppm), sphingomyelin (−0.09 ppm), lysophosphatidylcholine (−0.28 ppm), phosphatidylinositol (−0.30 ppm), alkylacylglycerophosphorylcholine (−0.78 ppm), choline plasmalogen (−0.80 ppm), and phosphatidylcholine (−0.84 ppm). Diabetic rats showed that phosphatidylcholine was significantly elevated p > 0.05, and ethanolamine plasmalogen and choline plasmalogen were significantly lower when compared with both control and insulin treated rats. The choline ratio (choline-containing phospholipids over noncholine phospholipids) was significantly elevated in the diabetic group, when compared with both control and insulin-treated groups. The ethanolamine ratio (ethanolamine-containing phospholipids over nonethanolamine phospholipids) and the ratio of the ethanolamine ratio over the choline ratio, was significantly elevated in the control and the insulin-treated groups when compared with the diabetic group. The presence of phosphatidic acid and the significance in phosphatidylcholine and ethanolamine plasmalogen, suggested that insulin had a role in the phosphatidylcholine metabolism in the rat nerve.     

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

Tissue polyunsaturated fatty acids and a digestive phospholipase A2 in the primary screwworm,Cochliomyia hominivorax

We report on the presence of arachidonic acid in larval and adult tissues of the primary screwworm, Cochliomyia hominivorax and of the secondary screwworm, C. macellaria. Arachidonic acid is present in the phospholipids of whole animal extracts of both species. This fatty acid appears to be accumulated during the larval stages, because proportions of arachidonic acid were higher in adults than in larvae. These insects probably obtain the arachidonic acid from dietary phospholipids. We also report on a phospholipase A₂ activity in midgut preparations from third instars of the primary screwworm. Phospholipase A₂ is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids to release essential fatty acids. The screwworm enzyme is similar to mammalian digestive phospholipase A₂s because it depends on calcium for high catalytic activity, it is sensitive to the site-specific inhibitor oleyloxyethylphosphorylcholine, and it interacts with heparin. We further characterized the screwworm midgut phospholipase A₂ by altering the reaction conditions, including reaction time, radioactive substrate concentration, protein concentration, pH and temperature. We speculate that the biological significance of this enzyme relates to acquiring essential fatty acids, including arachidonic acid, from dietary phospholipids.     

Acylation of monoacylglycerophosphoethanolamine in the inner and outer membranes of the envelope of an Escherichia coli K12 strain and its phospholipase A-deficient mutant

The site of the Escherichia coli envelope of the conversion of 1-acylglycero-3-phosphoethanolamine to diacylglycerophosphoethanolamine was explored, using two K12 strains with a wild-type phospholipid-degradative apparatus and a K12 mutant lacking detectable phospholipase A₁ and A₂ activity.Experiments with various radioactively labeled substrates show that acylation by crude envelope preparations as well as isolated inner and outer membranes of parent and mutant strains involves neither exogenous fatty acids nor a transacylation reaction with added monoacylglycerophosphoethanolamine. Furthermore, acylation exhibits no absolute requirement for added ATP and coenzyme A.Specific activity of acylating activity is the same in inner membrane preparations of parent and mutant strain and in outer membrane preparations of the mutant deficient in phospholipase A. Although clearly evident, net diacylglycerophosphoethanolamine formation by outer membranes of the parent strain, however, was about 6-fold less. This lower conversion may be attributed to activation during incubation of phospholipases A within the outer membrane, resulting in breakdown of the diacylcompound formed.Reacylation of lysophospholipids formed in the E. coli envelope by the action of endogenous or exogenous phospholipases A provides the organism with the potential of biochemically inexpensive repair and modification of the envelope phospholipids. Moreover, major phospholipids hydrolyzed in the outer membrane of E. coli can be resynthesized in the same location, without need for the transport of the products of hydrolysis to the lipid biosynthetic apparatus associated with the cytoplasmic membrane.     

Seasonal composition of lipids, fatty acids, and sterols in the edible red alga Grateloupia turuturu

Composition of lipids, sterols, fatty acids (FA), and phospholipids in the edible Rhodophyta Grateloupia turuturu from Britanny, France, was investigated over four seasons in order to identify compounds with potential benefits in health and nutrition. The lipid content was found to vary from 3.3 to 4.1 % dry weight. No marked variations were observed for glycolipids accounting for 42.3–46.8 %, whereas neutral lipids and phospholipids fluctuated from 20.1 % (summer) to 41.8 % (winter), and 11.2 % (winter) to 33.4 % (summer), respectively. Polyunsaturated FA of the total lipids were found from 20.4 % (winter) to 31.1 % (summer), including 20:5 ω3 acid as the major one (up to 16.3 % in summer). Phosphatidylcholine (20.0–43.7 %) and phosphatidylserine (24.6–37.5 %) were the dominant phospholipids in all seasons. Compounds of interest were identified in minor amounts such as squalene, α-tocopherol, phytonadione (vitamin K₁), cholesteryl formate, cholest-4-en-3-one, and cholesta-4,6-dien-3-one. Cholesterol was the major sterol with a lower content in spring and summer.     

Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations

The action of phospholipase C (Bacillus cereus) on the phospholipids of myelin sheath preparations has been investigated. With freshly isolated bovine brain myelin about 40% of the total phospholipid could be hydrolyzed by this enzyme. With bovine spinal cord myelin the phospholipid seemed more resistant to attack whereas the opposite was the case with myelin from guinea-pig brain or rat brain. With fresh bovine brain myelin, phosphatidylcholine and the ethanolamine-containing phospholipids were the main targets for the enzyme with lesser extents of hydrolysis occurring with phosphatidylserine and sphingomyelin. The effect of exposing bovine brain myelin to structural perturbants prior to enzyme digestion indicated that trypsin pretreatment had no significant effect, whereas marked enhancement of the extent of phospholipid hydrolysis occurred following lyophilization + rehydration, or pretreatment of myelin with HCl, Triton TX-100/ammonium acetate or deoxycholate. The effect of myelin pretreatment on the degradation of the individual phospholipid classes was also studied.     

Resolution and Reconstitution of a Mammalian Membrane

The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F ₁, Mg⁺⁺, phospholipids, and F_c. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c ₁, cytochrome c, cytochrome oxidase, phospholipids and Q ₁₀. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin.     

Increased membrane surface positive charge and altered membrane fluidity leads to cationic antimicrobial peptide resistance in Enterococcus faecalis

To understand the role of cell membrane phospholipids during resistance development to cationic antimicrobial peptides (CAMPs) in Enterococcus faecalis, gradual dose-dependent single exposure pediocin-resistant (Ped^r) mutants of E. faecalis (Efv2.1, Efv3.1, Efv3.2, Efv4.1, Efv4.2, Efv5.1, Efv5.2 and Efv5.3), conferring simultaneous resistance to other CAMPs, selected in previous study were characterized for cell membrane phospholipid head-groups and fatty acid composition. The involvement of phospholipids in resistance acquisition was confirmed by in vitro colorimetric assay using PDA (polydiacetylene)-biomimetic membranes. Estimation of ratio of amino-containing phospholipids to amino-lacking phospholipids suggests that phospholipids in cell membrane of Ped^r mutants loose anionic character. At moderate level of resistance, the cell-membrane becomes neutralized while at further higher level of resistance, the cell-surface acquired positive charge. Increased expression of mprF gene (responsible for lysinylation of phospholipids) was also observed on acquiring resistance to pediocin in Ped^r E. faecalis. Decreased level of branched chain fatty acids in Ped^r mutants might have contributed in enhancing rigidification of cell membrane and contributing towards resistance. The interaction of pediocin with PDA-biomimetic membranes prepared from wild-type and Ped^r mutants was monitored by measuring percent colorimetric response (%CR). Increased %CR of pediocin against PDA-biomimetic membranes prepared from Ped^r mutants confirmed that cell membrane phospholipids are involved in the interactions of pore formation by CAMPs. There was a direct linear relationship between percent colorimetric response and IC₅₀ of CAMPs for wild-type and Ped^r mutants. This relationship further reveals that in vitro colorimetric assay can be used effectively for quantification of resistance to CAMPs.     

Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity

Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (<5% of total protein S) binds with high affinity to phospholipid bilayers (K(d) < 1 nM). The remaining protein S (>95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (<5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.