Gel phase preference of ganglioside GM1 at low concentration in two-component, two-phase phosphatidylcholine bilayers depends upon the ceramide moiety.

In two-component phosphatidylcholine bilayers with coexisting liquid and P beta' gel phases, the distribution between phases of low concentrations of glycosphingolipids can be determined by freeze-etch electron microscopy after labeling the glycolipid with a suitable protein. We have found that the distribution depends upon the glycosphingolipid species (Rock, P. et al., (1991) Biochemistry 30, 19-25). Using this technique with cholera toxin as the protein label and bilayers formed from dipalmitoyl- and dielaidoylphosphatidylcholine (1:1) containing < 1 mol% GM1, we have studied the distribution of a family of GM1 homologues differing in the acyl chain and sphingoid base moieties. The GM1 preference for the P beta' ripple phase decreases with decreasing acyl chain length and increasing unsaturation. GM1 with either a C18:1 or C20:1 sphingoid base shows similar distributions in liquid and gel phases. When the molecules are preferentially found in the P beta' phase, they are positioned along unique loci in both A and A/2 forms of the ripple structure. This localization and acyl chain dependence reflect the volume, shape and localization of molecular packing defects in the P beta' phase. The ganglioside inclusions stabilize the P beta' phase and form compositional domains of unique topography.

New approach for atomic force microscopy of membrane proteins. The imaging of cholera toxin.

We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer. A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol % ganglioside (GM1) was transferred onto the surface of mica from a Langmuir trough. Cholera toxin, both the B-subunit and the complete molecular randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm. The pentameric structure of the B-subunit oligomers was well resolved. This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

Ganglioside GM1 and asialo-GM1 at low concentration are preferentially incorporated into the gel phase in two-component, two-phase phosphatidylcholine bilayers.

Multilamellar liposomes composed of 1:1 dielaidoylphosphatidylcholine: dipalmitoylphosphatidylcholine at 20 degrees C contain laterally separated gel and liquid-crystalline phases that can be identified by electron microscopy in freeze-etch replicas on the basis of their distinctive morphology. Visualization of marker proteins that specifically bind to glycosphingolipids included in these liposomes has revealed that, at 1 mol % or less, the ganglioside GM1 and the neutral asialo-GM1 derived from it are localized within the gel-phase regions exclusively. Increasing the mole fraction of the glycosphingolipids results in the appearance of marker in the fluid-phase regions. Another neutral glycosphingolipid, Forssman, does not display a phase preference and is found in both phases at a low mole percent. The phase preference of these three glycosphingolipids depends primarily upon interactions between the hydrophobic moieties of these molecules and the matrix phosphatidylcholines.

Organization of glycosphingolipids in phosphatidylcholine bilayers: use of antibody molecules and Fab fragments as morphologic markers.

The techniques of ultrafast freezing and freeze-etch electron microscopy have been successfully employed to visualize IgG molecules and Fab fragments specifically bound to the neutral glycosphingolipids Forssman and asialo-GM1 incorporated into phosphatidylcholine liposomes. Monovalent Fab is the superior marker because of its small size and because it does not cause liposomal aggregation with concomitant glycolipid reorganization. Analysis of Fab labeling of liposomes containing these neutral glycosphingolipids leads to the conclusion that the Forssman glycosphingolipid is dispersed in clusters of not more than several molecules when present at low mole fraction in fluid-phase 1-palmitoyl-2-oleoylphosphatidylcholine liposomes. In contrast to this, asialo-GM1 under the same conditions is present in clusters of about 15 molecules in this phospholipid matrix.

Persistence at low temperature of the P beta' ripple in dipalmitoylphosphatidylcholine multilamellar vesicles containing either glycosphingolipids or cholesterol.

The disappearance and reappearance of the P beta' ripple in multilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) has been examined by freeze-etch electron microscopy. The presence of less than 10 mol% of various glycosphingolipids or cholesterol in the liposomes markedly increases the time required for ripple disappearance when the vesicles are cooled from 38 degrees C to 30 degrees C, as compared to the pure phospholipid. Once the ripples have begun to disappear in the two-component vesicles, they do not uniformly reappear until the system is heated above the main transition of DPPC and allowed to cool into the pretransition region. These results suggest that the long time for ripple disappearance in the two-component systems reflects a slow molecular reorganization process which occurs when the systems are forced to change from the P beta' gel to the L beta' gel by a temperature downshift.

Electron microscopic identification of exposed plasmin epitopes in alpha 2-macroglobulin-plasmin complex using monoclonal antibody-colloidal gold adducts.

Monoclonal antibodies 10-F-1, directed against the K4 region of plasminogen, and 10-V-1, directed against the K1-3 region of plasminogen, were adducted to colloidal gold. These antibody-gold adducts bound specifically to alpha 2-macroglobulin (alpha 2M)-plasmin. Greater than 90% of the apparent binding was eliminated when alpha 2M-methylamine was substituted for the alpha 2M-plasmin. The plasmin epitope recognized by 10-F-1 was identified at the extreme pole of the alpha 2M-plasmin complex, suggesting that plasmin protrudes from the end of the hollow cylinder formed by alpha 2M. The complexes formed between alpha 2M-plasmin and 10-V-1 were indistinguishable from those formed with 10-F-1. This suggests that exposure of plasmin surface structure in alpha 2M-plasmin, while substantial, may be limited to the single region of the inhibitor. Evidence for ternary complexes containing one alpha 2M and two plasmin molecules was obtained in the form of antibody-gold bound at both poles of alpha 2M-plasmin. The fraction of alpha 2M-plasmin that associated with more than one antibody was small. The data presented here are considered in relation to current models of alpha 2M structure and function.

Polarity of the Forssman glycolipid in MDCK epithelial cells.

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.

Organization of ganglioside GM1 in phosphatidylcholine bilayers.

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

Organization of glycosphingolipids in bilayers and plasma membranes of mammalian cells.

The evidence presented in this review strongly suggests that, when present as a minor component in liquid crystalline phospholipid bilayers, neutral glycosphingolipids are segregated into compositional domains of small size dispersed in the matrix phospholipid. In many instances the glycosphingolipid in the dispersed domains is in the gel state. Because these domains are in the gel state, the individual molecules escape only very slowly from the surface of the bilayer, much more slowly than do the phospholipid components. There is as yet no direct evidence that this slow escape rate is a property of neutral glycolipids in biological membrane bilayers. If it is, however, then these molecules are well suited for their putative role as cell surface markers, a role that involves them in many important biological functions. There is evidence to suggest that molecules of this type are also present in a dispersed microdomain structure on the external surface of at least some mammalian cell plasma membranes. These small domains of glycolipids with their sugar residues projecting outward from the cell surface are much like a large membrane glycoprotein when viewed from the ambient medium near the cell surface. Thus, whether the sugar residues be of glycoprotein or glycolipid origin, they are localized in groups or patches on the external surface of the cell. One important consequence of this patch structure may be in the obvious effect on the free energy of binding a ligand to a patch. Whether the ligand is mono- or polyvalent, the roughly 2 M concentration of sugar in the surface patch will cause the apparent ligand binding free energy to be substantially larger than it would be for a single isolated sugar residue on the surface. In contrast to the neutral glycosphingolipids (and sulfatides, perhaps) the available information suggests that gangliosides are not localized in small domains in model systems and most probably not in biological membranes. Capping of this type of glycosphingolipid does appear to occur under certain circumstances. However, it is almost certain that capping is not an intrinsic property of ganglioside phospholipid-bilayer systems. Although at 37 degrees C gangliosides rapidly transfer from micelles to phospholipid vesicles and to cell membranes, nothing is known about the rates at which this class of molecules leave a phospholipid bilayer. Their known biological functions on the cell surface appear to require that they leave very slowly, if at all, as do the neutral glycosphingolipids. The glycosphingolipids are, by virtue of their polysaccharide moeity, a unique class of lipids and cell surface components.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of globoside and Forssman glycolipids on erythrocyte membranes.

Using the freeze-etch technique, the membrane localization of globoside, a principal glycolipid in human erythrocytes, and Forssman antigen, the chief glycolipid in sheep erythrocytes was evaluated using ferritin and colloidal gold as morphological markers for rabbit antibodies prepared against these glycolipids. Brief trypsinization of human red cell ghosts markedly aggregated intramembranous particles and permitted labeling of globoside, which appeared in a clustered arrangement. The aggregates of ferritin-anti-globoside differed from those of ferritin-wheat germ agglutinin, a label for glycophorin, which corresponded with the aggregates of intramembranous particles. Double-labeling of human trypsinized ghosts with anti-globoside/ Staphylococcal protein A-colloidal gold and ferritin-wheat germ agglutinin indicated that the patterns of labeling were different and that the aggregates of globoside did not bear a direct relationship to the intramembranous particles, which represent transmembrane proteins. Resealed sheep erythrocyte ghosts labeled with ferritin-conjugated rabbit anti-Forssman showed small clusters of Forssman glycolipid on the erythrocyte surface, which could be markedly aggregated with a second goat anti-rabbit antibody, indicating relative mobility of the small glycolipid domains. The distribution of ferritin-anti-Forssman label in sheep ghosts treated at pH 5.5 to aggregate intramembranous particles also did not show definite correspondence between intramembranous particles and the clusters of ferritin-anti-Forssman.

Organization of the glycosphingolipid asialo-GM1 in phosphatidylcholine bilayers.

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-GM1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-GM1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95-109 (1980)). Above the main phase transition temperature, asialo-GM1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Fusion of dipalmitoylphosphatidylcholine vesicles at 4 degrees C.

Small sonicated dipalmitoylphosphatidylcholine vesicles when incubated at 4 degrees C and high concentrations are shown to fuse completely to vesicles about 700-A diameter in 7 days, and these further fuse to about 950 A diameter vesicles after 3-4 weeks. The 950 A diameter vesicles are spherical, homogeneous, mostly unilamellar, have an internal aqueous space about 10 times that of small vesicles, and are stable for at least 6 months. The 950-A vesicles are characterized by agarose gel chromatography, freeze-fracturing electron microscopy, trapped volume measurements, differential scanning calorimetry, and diphenylhexatriene fluorescence polarization.

Fusion of dipalmitoylphosphatidylcholine vesicles.

Small unilamellar dipalmitoylphosphatidylcholine vesicles formed by sonication are shown to fuse spontaneously below the phase transition temperature. The ultimate fusion products are unilamellar vesicles about 700 A in diameter, which are stable and provide an intact ionic permeation barrier either above or below the phase transition. The fused vesicles have been characterized by gel chromatography, trapped volume, 31P nuclear magnetic resonance, and negative stain and freeze-fracture electron microscopy.

Staging laparotomy in Hodgkin's disease.

Staging laparotomy was performed at the University of Virginia Medical Center on 111 patients with Hodgkin's disease. The operation included multiple liver and lymph node biopsies and, excepting three patients, splenectomy. The histopathology was reviewed and the 111 patients were classified as follows: nodular sclerosis, 74; mixed cellularity, 28; lymphocyte predominance, 7; and undetermined, 2. There were no deaths. Wound, pulmonary or urinary tract complications occurred in 11 patients. One case of postoperative thrombophlebitis occurred and in another case small bowel obstruction developed, and resolved without reoperation. The pathologic stage (PS) following laparotomy was unchanged from the clinical stage (CS) in 64%, reduced in 20%, and advanced in 16%. The therapy, however, was altered in 38% of the patients. Lymphangiography in 103 patients was interpreted as showing lymph node involvement in 38, equivocal involvement in 11, and no involvement in 54. Among the 92 examinations reported as either positive or negative, 77% were confirmed histopathologically, 21% were falsely positive, and 2% were falsely negative. The spleen was positive for Hodgkin's disease in 39% of cases, and in these patients with positive spleens there was no reason to suspect intra-abdominal involvement preoperatively in 21%.

Extended field radiation therapy in Hodgkin's disease: analysis of failures.

A retrospective analysis of 116 primary cases of stage I, II and III-A Hodgkin's disease demonstrated 27 failures, fourteen confirmed by biopsy. Twenty of the 27 (74%) were in lymph node areas only and seven had extranodal extensions. The most frequent site of failure was the hilar nodes and contiguous lung. The majority of failures (78%) occurred within 30 months of treatment and the cause of failure determined in 23 (85%), sixteen of which were due to technical errors of irradiation. Analysis of the dosimetry in the mediastinal, hilar nodes and contiguous lung failures revealed three factors which may have contributed to a low dose. These factors are: 1) the equivalent square, 2) off-axis beam diminution, and 3) the anteroposterior dose profile. Combined radiation therapy and chemotherapy was more effective than either modality alone in the treatment of these failures.

Development of sarcoplasmic reticulum in cultured chicken muscle.

The development of sarcoplasmic reticulum membranes was studied in vivo and in tissue culture in chicken pectoralis muscle cells. The concentration of the calcium- and magnesium-activated ATPase measured by selective labeling of the enzyme with [32P]ATP in whole muscle homogenates was found to increase in developing chicken pectoralis muscle in vivo from 0.01 nmol/mg of protein in 12-day embryos to 0.3 to 0.4 nmol/mg of protein in 1-month-old chicks, where it constitutes about 3% of the total protein content of muscle. In cultured muscle cells the concentration of calcium-sensitive phosphoprotein increased from 0.015 nmol/mg of protein at 2 days to 0.04 to 0.05 nmol/mg of protein after 5 days of culture. This amount represents about 0.5% of the protein content of the muscle cells. The accumulation of Ca2+ transport ATPase began during fusion and continued with a linear rate during 8 days of culture. The density of 75 A intramembranous particles seen by freeze-etch electron microscopy on fracture faces of sarcoplasmic reticulum membranes is about 4,000/mum2 in adult chick pectoralis muscle but only 400/mum2 in cultured muscle cells in rough proportion to the concentration of Ca2+-sensitive phosphoprotein. The Ca2+, Na+, and K+ concentration of the medium and addition of ouabain, caffeine, or the calcium ionophores A23187 and X537A sharply influence the concentration of calcium transport ATPase in cultured muscle cells, parallel with their effect upon cell fusion and growth. These observations are consistent with the proposition that the gene expression leading to the accumulation of Ca2+ transport ATPase during development in culture may be regulated by intracellular ion concentrations.

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes.

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.

Gamma heavy chain disease: rapid, sustained response to cyclophosphamide and prednisone.

A patient, CAL, with gamma heavy chain disease is presented who has had a complete remission lasting over 2 yr with combination chemotherapy consisting of pulsatile cyclophosphamide and prednisone. The patient exhibited many features of an atuoimmune process including a vasculitis, low serum complement levels, a positive antiglobulin (Coombs) test, Raynaud's phenomenon, and keratoconjunctivitis sicca. The CAL paraprotein was found to have several previously undescribed characteristics. It reacted with antisera to Fd, Fab, and Fab', suggesting that most of the Fd portion of the molecule was intace. CAL protein consists of two polypeptide chains of molecular weight 49,000 covalently linked to form a dimer of 95,000 molecular weight. The covalent linkage suggests that the hinge region of this gamma heavy chain is intact.