Separation and localization of cell wall layers of a gram-negative bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.     

Distribution of lipopolysaccharide and the detection of a new subfraction in the cell envelope of a marine pseudomonad.

The three outer layers of the cell envelope of marine pseudomonad B-16, the loosely bound outer layer, the outer membrane, and the periplasmic space layer, are the only ones containing appreciable amounts of both lipid and carbohydrate. These layers and a fraction released into the medium during growth of the cells were examined for the presence of common antigens by double immunodiffusion using anti-whole serum. Each of the layers, the medium fraction, and lipopolysaccharide (LPS) isolated from the organism were shown to contain two or more diffusible components showing reactions of identity. Thus LPS is found in each of the three outer layers of the cell envelope of this gram-negative bacterium. The periplasmic space layer was found to contain a fraction accounting for 20% of the dry weight of the layer, which was sedimentable at 30,000 x g and contained lipid, protein, and carbohydrate. Double-immunodiffusion tests indicated that the fraction contained at least one of the two antigens present in isolated LPS. A particulate material was released by the cells during growth which gave a positive test for 2-keto-3-deoxyoctulosonic acid and cross-reacted serologically with LPS.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

Isolation and chemical characterization of outer envelope of Leptospira pomona.

Cells of Leptospira interrogans serotype pomona harvested from a chemically defined medium were resuspended in 0.01 M phosphate buffer of pH 7.3. Electron microscopy showed that 90 min of exposure effectively ruptured the outer envelope, freeing it from the cells as small flakes. Both zonal centrifugation in sucrose gradients and centrifugation in isopycnic KBr and CsCl gradients could be used to separate the outer envelope from the axial filaments and protoplasmic cylinders. The latter method resulting in higher yields of purified envelope with the particular protocols used. Thin sections of isolated outer envelope showed the same trilaminar structure seen in sections of intact cells. The outer layers were 1.5 nm thick and appeared as single layers of electron-dense particles. The central electron-transparent layer was 2.0-2.5 nm thick and appeared structureless. The gross chemical composition of the purified outer envelope was 47% protein, 27% carbohydrate, and 23% lipid. Colorimetric carbohydrate determinations revealed hexose, pentose, and 6-deoxyhexose; hexosamine was identified during amino acid analysis. Muramic acid, heptose, and 2-keto-3-deoxyoctonate were not detected. Thin-layer chromatography revealed only polar lipids, about 98% phosphatidylethanolamine and 2% lysophosphatidylethanolamine. Fatty acids identified by gas-liquid chromatography were octadecanoic, octadecenoic, hexadecanoic, and hexadecenoic. Amino acid analysis revealed 17 amino acids, histidine and glutamic acid being most abundant. The outer envelope was interpreted to be comparable with the outer double-track layer found in the cell covering of gram-negative eubacteria.     

Heterogeneity and Distribution of Lipopolysaccharide in the Cell Wall of a Gram-Negative Marine Bacterium

Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and III, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS III, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I.     

Isolation, Composition, and Structure of Membrane of Listeria monocytogenes

The plasma membrane of Listeria monocytogenes strain 42 was prepared by osmotic lysis of protoplasts with tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.2, containing MgCl₂ and glucose, followed by washing with NaCl and MgCl₂ in Tris buffer. Electron microscopy showed that the preparation was not contaminated with cytoplasmic material. The membrane preparation was composed of 55 to 60% protein, 1.5% ribonucleic acid, 0.1% deoxyribonucleic acid, 1.3 to 2.3% carbohydrate, 0.17 to 0.38% amino sugar, 0.2 to 0.4% rhamnose, 3.5 to 4.0% phosphorus, 10.5 to 12.0% nitrogen, and 30 to 35% lipid. Amino acid composition of the washed membrane showed some variation from that of the whole cells. Sulfur-containing amino acids were not present in the membrane hydrolysate. The membrane carbohydrate contained glucose, galactose, ribose, and arabinose. The membrane lipid was 80 to 85% phospholipid and 15 to 20% neutral lipid. The lipid contained 2.3 to 3.0% phosphorus, 2.5 to 3.0% carbohydrate, and a very small amount of nitrogen (0.2 to 0.3%). The phospholipid was of the phosphatidyl glycerol type. Electron micrographs of the washed membrane showed three layers. The outer and inner layers varied in thickness from 25 to 37 A and the middle layer from 20 to 25 A. The total thickness varied between 85 and 100 A. These preparations contained many vesicles which stained heavily with lead citrate. Some vesicles were also attached to the protoplast ghosts in the form of extrusions or intrusions, or both. Membrane preparations obtained by lysis of protoplasts in the absence of MgCl₂ were fragmented and contained less lipid (20 to 22%) and ribonucleic acid (0.3 to 0.5%) than preparations prepared with MgCl₂.     

Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp

Chemical analysis of fractions of the cell envelope of Acinetobacter sp. strain MJT/F5/199A, prepared by breakage in the French press and removal of plasma membranes, followed by sequential treatment with lysozyme and with papain, confirmed the existence of layers previously identified by electron microscopy. Outside the plasma membrane and periplasmic space, the envelope is composed of (i) a peptidoglycan-containing dense layer, (ii) an intermediate layer, (iii) a lipopolysaccharide-containing outer membrane, and (iv) an ordered array of protein subunits. A small amount of carbohydrate (3%) is found associated with protein in the fraction containing both the surface subunits and the intermediate layer. The papain-treated outer membranes contain 67% protein, 24% lipid, together with 11% lipopolysaccharide, and about 6% of non-lipopolysaccharide hexosamine. Lipid is located only in the papain-treated outer-membrane and is mainly phospholipid: 29% phosphatidyl glycerol, 30% phosphatidyl ethanolamine, and 40% cardiolipin. The principal fatty acid is C(18:1). Significant amounts of alcohols(16:1) and alcohols(18:1), which are found in Acinetobacter waxes, were recovered from the outer membrane.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Protein and Carbohydrate Composition of the Cell Envelope of Halobacterium salinarium

The isolated cell envelope of Halobacterium salinarium strain 1 contained 15 to 20 proteins that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All but one of these proteins had molecular weights of 130,000 or less and together accounted for 50 to 60% of the total envelope protein. The remaining 40 to 50% of the envelope protein was accounted for by a single protein with an apparent molecular weight of approximately 194,000 that stained for carbohydrate with periodate-Schiff reagent. The proteolytic enzymes trypsin and Pronase were used to show that the carbohydrate is covalently bound to the protein. Separation of amino sugar- and hexose-containing tryptic peptides by gel filtration indicated that all of the nonlipid carbohydrate of the cell envelope is covalently bound to protein. The results of partial purification by phenol extraction indicated that both the amino sugar and hexose are bound to the 194,000-molecular-weight protein. Exposure of isolated cell envelopes to low salt concentration resulted in solubilization of a majority of the envelope proteins. A relatively small number of proteins, including the high-molecular-weight, carbohydrate-containing protein, remained bound to the sedimentable cell membrane fraction.     

Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda).

Histochemical and ultrastructural studies were made on the metacercarial cyst of Echinostoma revolutum obtained from the kidney of experimentally infected Physa and Lymnaea snails. Ultrastructural studies revealed three cyst walls, an outer, middle and inner. The outer wall was more electron-dense than the middle, and contained coarser granules than those found in the middle layer. The inner wall was lamellated and contained membranous whorls. Collagenous fibers presumably of host origin surrounded the outer cyst wall. The outer and middle cyst walls stained identically with all histochemical procedures used. These walls contained acid mucopolysaccharides and glycoprotein, whereas the inner cyst wall contained glycoprotein. All cyst walls stained positively with a variety of protein stains.     

Glycosaminoglycans and glycoproteins isolated from rabbit femur.

1. Complex carbohydrate fractions were extracted successively with 40% aqueous EDTA (pH 7.4) and 6M urea (PH 7.8) FROM ACETONE-DRIED bone powder of rabbit femur. 2. The carbohydrate fraction extracted with EDTA (E=Fr) was separated into five fractions,D1approximatelyD5 by DEAE-Dephadex A-50 column chromatography. Chemical and infrared spectral analyses, and enzymatic digestion indicate that D2 contained lessacidic glycoprotein, D3 contained sialoglycoprotein, D4 contained a low sulfated proteokeratan sulfate-like substance, and d5 contained glycoprotein-bound chondroitin sulfate A plus protein-free chondroitin sulfate A. 3. Two fractions, HU-D1 and HU-D2, were isolated from the carbohydrate fraction extracted with urea (HU-Fr) by successive digestion with collagenase [EC 3.4.99.5] and pronase, followed by gel-filtration on Sephadex G-100 and then DEAE-Sephadex A-50 column chromatography. HU-D1 and HU-D2 contained a low sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfate A, respectively. 4. The present findings indicate that rabbit femur contains low sulfated proteokeratan sulfate-like substances with varying sulfate contents and glycoprotein-bound chondroitin sulfate A as the principal glycosaminoglycans. The macromolecules bound more tightly to the tissue contain much more sulfate than the corresponding loosely bound ones.     

Cell surface proteoglycans as a negative modulator in concanavalin a-mediated agglutination of hepatoma cells

Proteoglycan (heparan sulfate-protein conjugate) was solubilized with 8 M urea from rat liver plasma membranes after enzymic (RNAase, neuraminidase) treatments and extensively purified by chromatography and gel filtration. The final products gave an average ratio of hexuronate to protein (weight) of approx. 1.5, contained hexosamine equimolar to hexuronate and were sensitive to β-elimination (the molecular weight being reduced from 20 · 10⁴ to 3 · 10⁴ (gel filtration)).The proteoglycan fraction, when added to trypsinized and untrypsinized ascites hepatoma (AH-130F(N)) cells, inhibited the concanavalin A-mediated agglutination of the cells. However, the alkali-treated proteoglycan (β-elimination) or acid mucopolysaccharide fraction prepared from liver plasma membranes by papain digestion were less effective, and a reference preparation of heparan sulfate was almost ineffective. It was confirmed that significant amounts of proteoglycan labelled with ³⁵SO₄^2? were firmly bound to or taken up by the trypsinized ascites hepatoma cells.These results together with the sensitization of lectin-mediated agglutination by mild protease treatment of cells suggest that cell surface proteoglycans may act as a negative modulator in the lectin-mediated agglutination of cells.     

The envelope of Micrococcus radiodurans: isolation, purification, and preliminary analysis of the wall layers

Two methods are presented that separate the complex envelope of Micrococcus radiodurans, strain Sark, into its constituent layers. The first involved treating whole cells with 0.025 M Tris buffer (pH 7.5) containing 2 mM of calcium and 3 mM of magnesium, resulting in the degradation of an intermediate ('compartmentalized') layer and consequent sloughing of the outer subunit and interior layers to form vesicles. This treatment also appears to show that the interior layer may be connected with the peptidoglycan-containing 'holey' layer. The second method involves treating whole cells with benzene followed by sonication; the results suggested that this treatment only released the outer layers from the 'compartmentalized' layer and did not degrade layers. Following benzene treatment, digestion of the 'compartmentalized' layer with cold sodium dodecyl sulfate (SDS) released the 'holey' layer. Electrophoretic analysis of some of the isolated layer preparations suggested that the subunit layer consisted of three major proteins of 90 000, 92 000, and 94 000 molecular weight, one minor protein of 100 000, a small amount of carbohydrate associated with the 94 000 protein, and a small amount of a 55 000 lipoprotein. The interior layer contained at least 10 proteins and may be attached to the peptidoglycan-containing 'holey' layer by means of the 55 000 lipoprotein.     

EVIDENCE THAT THE MAJOR PROTEIN IN RAT SCIATIC NERVE MYELIN IS A GLYCOPROTEIN

Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid-Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [³H]leucine and [¹⁴C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [¹⁴C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.     

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

Isolation and characterization of plasma-membrane glycoproteins from pig epidermis

1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000–80000. 2. A large proportion of the marker enzymes, the d-[³H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A–Sepharose 4B and lentil lectin–Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) α-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[³H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[³H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.     

Expression pattern of Glycoconjugates in Rat Retina as Analysed by Lectin Histochemistry

The present study sought to characterize the expression and distribution of complex glycoconjugates in the rat retina by lectin histochemistry, using a panel of 21 different lectins with different carbohydrate specificities. Paraffin sections of Carnoy-fixed Sprague–Dawley rat eyes were stained with various biotinylated lectins, followed by the streptavidin-peroxidase and glucose oxidase–diaminobenzidine–nickel staining procedures. The results showed that the retinal pigment epithelium was stained intensely with LCA, Jacalin, WFA, S-WGA, PWA, DSA, UEA-I, LTA and PHA-E, suggesting that this epithelium contained glycoconjugates with α-Man, α-Glc, α-Gal/GalNAc, β-GalNAc, α-Fuc, NeuAc and other oligosaccharide residues. The outer and inner segments of the photoreceptor layer showed different lectin binding affinities. The outer segments reacted with S-WGA and GS-II, whereas the inner segments reacted with UEA-II, UEA-I, LTA and MAA, suggesting that the inner segments contained glycoconjugates rich in α-Fuc and NeuAc(α2,3)Gal residues. PNA labelled specifically the cones and could be used as a specific marker for these photoreceptors. RCA-I, WFA, S-WGA, DSA, MAA and PHA-E reacted with both the outer and inner plexiform layers. On the other hand, UEA-I and LTA specifically labelled the outer plexiform layer, while PNA labelled the inner plexiform layer. The retinal microglial cells were labelled specifically by GS-I-B₄ and SNA. Interestingly, we also observed that WFA bound specifically to Müller cells and could be used as a novel marker for this retinal glial cell. The capillaries and larger vessels in the retina and choriocapillaris reacted intensely with GS-I-B₄, RCA-I, S-WGA, PWA, DSA and PHA-E. No significant differences in lectin binding were observed in the microvessels at these two sites. In summary, the present study demonstrated the expression patterns of glycoconjugates in the rat retina and that certain lectins could be used as histochemical markers for specific structural and cellular components of the rat retina.     

Glycopeptide fractions prepared from purified central and peripheral rat myelin

Myelin was purified from rat brain and sciatic nerve after invivo labeling with [³H]fucose and [¹⁴C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 μg of neutral sugar and 0.59 μg of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 μg of neutral sugar and 3.8 μg of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and $\text{N-}\text{acetylglucosamine}$. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin in glycosylated while the glycoproteins in purified central myelin are quantitatevely minor components.