Incorporation of N-Acetylmannosamine into Rat Brain Subcellular Gangliosides: Effect of Pentylenetetrazol-Induced Convulsions on Brain Gangliosides1

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N-acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides G_T1b and G_Q1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, G_T1b and G_Q1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane-bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water-soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin-layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin-layer chromatogram.     

Procedure for isolation of gangliosides in high yield and purity: simultaneous isolation of neutral glycosphingolipids

While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.     

Molecular parameters of gangliosides in monolayers: comparative evaluation of suitable purification procedures

The molecular area, collapse pressure, and surface potential of gangliosides obtained by different methods were systematically compared in monolayers at the air-water interface. Different values of these parameters are obtained depending on the purification procedure employed for the isolation of pure gangliosides. This is due to impurities (such as peptidaceous material) that remain in different amounts in the various preparations and that modify the ganglioside surface behavior. Routine purity checking by HPTLC analysis of gangliosides usually fails to reveal these impurities. On the other hand, even if the monolayer technique cannot identify the nature or amount of contaminants, it is extremely sensitive to reveal alterations of the surface molecular parameters caused by relatively small amounts of other components coextracted with the ganglioside or adventitiously introduced with the solvents or subphases employed. This is a serious problem for the obtention of correct and reproducible values of such important parameters as the molecular area of gangliosides, their electrostatic potential in oriented interfaces, and their interactions with other lipids and proteins. A procedure leading to consistent molecular parameters that remain reproducible after several repurification cycles is to perform an alkaline treatment on previously purified gangliosides species with NaOH, this is followed by dialysis against bidistilled water, rechromatography on DEAE-Sephadex A25, silicic acid or Iatrobeads, and Sephadex LH-20 columns; repurified gangliosides are stored in chloroform-methanol-0.01 M NaOH (60:30:4.5).     

On the loss of gangliosides by dialysis

The Gangliosides represent a family of sialic acidcontaining sphingoglycolipids which have ceramide (N-acyl-sphingosine) as the basic hydrophobic portion (Svennerholm , 1972). A heteropolysaccharide is glycosidically linked through the primary hydroxyl group of ceramide and the aldehyde of glucose. There are usually three or four different carbohydrates and up to a total of seven in the chain. The isolated gangliosides are soluble both in organic and aqueous solvents presumably because of their content of both hydrophobic and hydrophilic groups. The most commonly employed extraction procedure is based upon their solubility in chloroform-methanol (2: 1, v/v) and subsequent partitioning into an aqueous methanol phase (FOLCH, LEES and SLOANE-STANLEY, 1957). This methanolic solution is then dialysed against water to remove small molecular-weight contaminants. This paper reports the loss of gangliosides upon dialysis when they are present at low concentrations. Gangliosides were prepared from fresh calf brain as previously described (Kanfer , 1969) and contained 25-25% GMl, as judged by quantitative thin-layer chromatography (Suzuki , 1964). GM2 was prepared from this material by treatment with neuraminidase as previously described (Kolodny , Brady , Quirk and Kanfer , 1970) and purified by the procedure of Winterbourne (1971). GM2 was isolated from brain tissue of a Tay-Sachs child provided by Dr. B. Volk. Thin-layer chromatography was carried out on Analtech Silica Gel G plates (Analtech Co., Wilmington, Del.) with chloroformmethanol-2.5 N NH₄OH (60 : 30 : 8, by vol.) as developing solvent. Total sialic acid was quantified by the resorcinol procedure according to Suzuki (1964). Duplicate known quantities of gangliosides were dissolved in 2 ml of methanol 41 M KCI(1 : 1, v/v) and dialysed overnight in untreated Visking tubing (0.22-in. dia.; Fisher Scientific Co., Pittsburgh, Pa.; Cat. No. 1000) against a 500-fold vol. of distilled water at 4°C. The contents of the sacs were removed and the total volume was measured; a 10-1 5 per cent increase in volume was usually observed. Portions were analysed in duplicate for total sialic acid content, and the remainder was lyophilized for examination by TLC.     

Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment

A method for the detection of GM1b-type gangliosides in complex mixtures of gangliosides was developed. The procedure involves separation of gangliosides on high-performance thin-layer chromatography plates, fixation of the silica gel, treatment with neuraminidase from Vibrio cholerae in the absence of detergent, and incubation of the plates with GgOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies are used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolylphosphate. The procedure is capable of detecting as little as 30 ng of gangliosides. Gangliosides from murine T lymphocytes and from human brain served as examples. Besides GM1b, GD1 alpha is also detectable by this method, whereas the human brain gangliosides GM1a, GD1a, GD1b, and GT1b are not, because they are neuraminidase resistant. Since terminally sialylated gangliosides such as GM1b were described as virus receptors, and certain other terminally sialylated gangliosides are discussed as tumor markers, this method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small amounts of material are available.     

Simple method for large-scale isolation and purification of gangliosides by non-ionic adsorption chromatography

A type of non-ionic adsorption resin, X-5, was used in the isolation and purification of brain gangliosides. Hydrolysis with base treatment was carried out for the purpose of eliminating contaminants. The major advantages of the new procedure, compared to conventional methods, were the shorter separation time, higher loading capacity (80 micromol LBSA per gram resin), and recovery (98%) of separated ganglioside fractions with little solvent. It is a practical way for large-scale isolation and purification of gangliosides.     

Study of myelin purity in relation to axonal contaminants

Axonal remnants are considered a probable source of contamination of isolated myelin in view of the relatively tight axon-glial intercellular junction. Using the rabbit optic system to label specifically axonal components, we have found the levels of such contaminants to depend on the myelin isolation procedure, the tissue source, and the nature of the contaminant. A procedure employing repetitive treatments with EGTA was found to be highly effective in removing proline-labeled axonal proteins, the estimated upper limit of such contamination being approximately 0.6–1.2% of the myelin protein. The standard isolation procedure of Norton and Poduslo, supplemented with an additional discontinuous gradient step, proved equally effective in removing rapidly transported proteins from myelin isolated from the superior colliculus or lateral geniculate body. When the optic tract was the source, however, the EGTA procedure proved more effective in removing both rapidly and slowly transported proteins. Axonal gangliosides labeled with N-[ ³ H]acetylmannosamine were efficiently removed by both procedures, adding support to the proposition that gangliosides detected in isolated myelin are intrinsic to that membrane.     

Monoclonal antibody 18B8, which detects synapse-associated antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer)

By immunofluorescence, mouse monoclonal antibody 18B8 detects developmentally regulated antigens in chick neural retina. In older embryos and in adults these antigens are localized in discrete laminae within the inner and outer synaptic layers. The antibody binds to several gangliosides that undergo both qualitative and quantitative changes during neuronal development (Grunwald, G.B., Fredman, P., Magnani, J.L., Trisler, D., Ginsburg, V., and Nirenberg, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4008-4012). The simplest of these gangliosides was isolated from lipid extracts of 10-day chick embryonic retinas by DEAE-Sepharose and silicic acid column chromatography. About 300 micrograms was obtained from 9.3 g (wet weight) of retina. The isolated ganglioside was identified as GT3 by enzymatic analysis and by a comparison of its properties with the authentic ganglioside. By immunostaining thin-layer chromatograms with antibody 18B8, GT3 was detected in gangliosides from human neural tissue including cerebellum, optic nerve, and spinal cord, but not in gangliosides from human liver, pancreas, small intestine, adrenals, thyroid, or erythrocytes. GT3 was also found in five of seven human melanoma cell lines.     

The purification of human serum 1 -antitrypsin by affinity chromatography on concanavalin A

α₁-Antitrypsin (α₁-AT) has been isolated from human serum by a two-step procedure which involves chromatography on DEAE-Sephadex followed by affinity chromatography on insolubilized concanavalin A. This protein appeared to be homogeneous when examined by electrophoresis on cellulose acetate and double immunodiffusion; minor contaminants, however, were detected by gel electrophoresis and immunoelectrophoresis. This procedure is readily adaptable to the large-scale purification of α₁-AT and should facilitate further studies on the physicochemical and biological properties of α₁-AT and its genetic variants.     

Fatty acids and long-chain bases of gangliosides of human gastrointestinal mucosa

The fatty acid and long-chain base composition of five major gangliosides from human stomach and small and large intestine mucosa were analyzed with gas chromatography. All the gangliosides greatly resembled each other in the fatty acid pattern. The main fatty acids were C_16:0, C_18:0 and C_24:0. No hydroxy fatty acids could be detected. In all the gangliosides 4-sphingenine was the predominant long-chain base (70–75%). About 15% of the long-chain bases had 20 carbon atoms in their chain. No trihydroxy long-chain bases could be detected.     

Quantitative analysis of brain gangliosides by high performance liquid chromatography of their perbenzoyl derivatives

This report describes a convenient, highly sensitive, and reproducible HPLC procedure for the quantitative analysis of gangliosides from brain tissues. The procedure involves the conversion of gangliosides to their perbenzoyl derivatives, isolation of the derivatives on a C18-reversed-phase cartridge, separation of the derivatives on a column (3-micron silica) maintained at an elevated temperature, and UV detection of the derivatives at 230 nm. The convenience of the procedure, its sensitivity, reproducibility, and application to the analysis of gangliosides from tissue sources make it the method of choice for ganglioside quantification in our laboratories. Three aspects of the procedure contribute to its convenience: reaction conditions that lead to single products, a convenient isolation procedure for the derivatives, and chromatographic conditions that provide resolution of the derivatives.     

Binary affinity chromatography for the purification of glycogen phosphorylase

A binary affinity chromatography medium was prepared and found to be useful for the purification and quantitative isolation of glycogen phosphorylase from rabbit skeletal muscle and liver. Glycogen is used as the binary ligand as it has affinity toward both the column matrix and the enzyme. Agarose beads derivatized with concanavalin A bound glycogen to the level of 35 mg/ml. The glycogen-impregnated beads were able to bind 9 mg/ml of phosphorylase a or b. The phosphorylase is tightly bound so that the column can be washed free of contaminants before quantitative elution of the phosphorylase by 2 M glucose, which releases the glycogen-phosphorylase complex. It appears that binary affinity chromatography may have general utility for the isolation and purification of enzymes and other specific binding agents.     

Densitometric quantification of brain gangliosides separated by two-dimensional thin layer chromatography

A procedure for accurate densitometric quantification of gangliosides separated by two-dimensional thin layer chromatography is reported. The procedure was set up employing 9 different pure gangliosides and was applied to the analysis of calf and pig brain gangliosides. Silica gel high performance thin layer plates, 10 × 10 cm. were two-dimensionally developed at 18–20 C with the following solvents: chloroform methanol 0.2% aqueous CaCl₂, 50/40/10 by volume, for the first run; n-propanol 17 M NH₄OH/water, 6/2/1 by volume for the second run. Ganglioside spots were visualized by spraying with an Ehrlich reagent, which is specific for sialic acid, and heating at 120 C for 15 min. The spots were quantified by sequential scanning densitometry, linear responses being obtained for ganglioside amounts on the plate ranging from 0.1 to 6 nmol as bound sialic acid. The reproducibility of densitometric responses resulted to be acceptable since the standard deviation values were lower than ± 15% of the mean values also for those ganglioside species contained in minor proportions. The ganglioside mixtures of calf and pig brain were resolved in about 20 spots. Of these 9 corresponded to gangliosides GM3, GM2, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b, which were identified with certainty and quantified. The identification of GM3 (carrying N-glycolylneuraminic acid), GD3, GD1a (carrying N-acetyl- and N-glycolyl-neuraminic acid) and GT1a was only tentative. All the other spots corresponded to unidentified gangliosides, some of them possibly new species.     

A modified procedure for the analysis of mixtures of tissue gangliosides

Abstract— A modified procedure is described for quantification of gangliosides in a mixture using thin-layer chromatography. Using this procedure, the gangliosides in human peripheral nerve were quantified, and compared with those in cerebral cortex and white matter.     

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin

A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K _i for neomycin (11 μM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A ₆₁₀/A ₂₈₀ ∼ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.     

Procedure for Isolation of the 20,000-Dalton Variant of Human Growth Hormone

Abstract

An isolation procedure for the 20,000-dalton variant of human growth hormone has been devised to improve the yield of the final product. The improvement involved elimination of cumbersome steps that decreased yield, and modification of chromatography on DEAE-cellu-lose to provide better separation of the variant from the major form of growth hormone.

We reported (1) an isolation procedure for the 20,000-dalton variant of human growth hormone (hGH_20K) (2) which provided quite homogeneous material but the yields were not optimum. The initial steps were cumbersome and losses resulted from them. In addition, there was the problem that in order to completely remove the major 22,000-dalton form of the hormone (hGH) during the final chromatography step (DEAE-cellulose), only the leading edge of the hGH_20k?peak could be used. The trailing part of the peak was always mixed with hGH and reworking this mixture resulted in losses. We have modified the procedure so that time-consuming steps were eliminated and the final chromatography step was improved so that now the hGH_20K can be separated from hGH by a single column.     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

Isolation of β-N-acetyl-d-hexosaminidases from lupin seed

A rapid procedure for the isolation of β-N-acetyl-d -hexosaminidase from lupin seed meal is described. This involves affinity chromatography of a seed extract on concanavalin A, followed by chromatography on DEAE-Sepharose. The purified enzyme was obtained in three forms, hexosaminidases A, B and B₁, capable of hydrolysing both p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside and p-nitrophenyl β-2-acetamido-2-deoxy-d-galactopyranoside. Enzyme A was relatively less active towards the galactosaminide substrate, than were the B forms of the enzyme.     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

Determination of ganglioside non-hydroxy fatty acid and long chain base by analysis of perbenzoylated derivatives

The non-hydroxy fatty acid and long chain base compositions from as little as 2.7 nmol of ganglioside were ascertained from perbenzoylated ganglioside derivatives. Non-hydroxy fatty acids were determined by mild alkaline methanolysis of the derivatives, followed by gas-liquid chromatography (GLC) of the methyl esters. N-acyl and N-benzoyl "gangliosides" that were generated by the methanolysis were hydrolyzed by a standard procedure that utilized aqueous acetonitrile-HCl, followed by high performance liquid chromatography (HPLC) determination of the biphenylcarbonyl derivatives with ultraviolet (UV) detection at 280 nm. A critical aspect of this procedure is a modified workup for the isolation of the biphenylcarbonyl derivatives which eliminates by-products that otherwise interfere with their separation by HPLC, especially when high sensitivity is required.