Structural characterization of a novel cholinergic neuron-specific ganglioside in bovine brain.

A new ganglioside antigen, termed Chol-1 alpha-b, recognized by cholinergic neuron-specific antibody, Chol-1 alpha, was isolated from bovine brain ganglioside mixture using Q-Sepharose. The yield was approximately 1.3 mg from 5 g of the total ganglioside. The chemical structure was characterized as a novel ganglioside by means of gas-liquid chromatography, a permethylation study, mild acid hydrolysis, thin layer chromatography-enzyme immunostaining, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ganglioside has the following unique structure. [formula: see text] When examined by thin layer chromatography immunostaining and enzyme-linked immunosorbant assays, this ganglioside has the most intense immunoreactivity with Chol-1 alpha antibody among bovine brain gangliosides. As combined with our previous results (Hirabayashi, Y., Hyogo, A., Nakao, T., Tsuchiya, K., Suzuki, Y., Matsumoto, M., Kon, K., and Ando, S. (1990) J. Biol. Chem. 265, 8144-8151; Ando, S., Hirabayashi, Y., Kon, K., Inagaki, F., Tate, S., and Whittaker, X. (1992) J. Biochem. (Tokyo), 111, 287-290), the present study indicates the occurrence of a new series of gangliosides containing N-acetylneuraminic acid residue attaching to N-acetylgalactosamine as cholinergic specific antigens.     

Isolation of two novel adenosine binding proteins from bovine brain

Adenosine plays many significant roles both as a metabolic precursor and cell communicator. This report describes the preliminary characterization of two adenosine binding proteins isolated from bovine brain membranes. By using N6-9-aminononane adenosine labeled Sepharose 4B two major affinity bound proteins were purified having apparent molecular weights of 16 and 35 kDa. Either or both of the proteins could be selectively eluted from the affinity column with N6-9-aminononane adenosine, adenosine, cAMP, AMP, ADP, ATP, R-/S-phenylisopropyladenosine and NAD(H). By contrast, no proteins were eluted with caffeine, adenine, deoxyadenosine, 2',3'-AMP, inosine, IMP, xanthine, XMP, GMP, GTP or 5'-N-ethylcarboxamideadenosine. The selectivity of elution and lack of apparent enzymatic activity suggests that these proteins are novel membrane bound adenosine binding proteins.     

Isolation of two glycolipid transfer proteins from bovine brain: reactivity toward gangliosides and neutral glycosphingolipids

Two glycolipid transfer proteins that catalyze the transfer of gangliosides and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts have been isolated from calf brain. Purification procedures included differential centrifugation, precipitation at pH 5.1, ammonium sulfate precipitation, and gel filtration on Sephadex G-50 and G-75. The final stage employed fast protein liquid chromatography (Mono S), producing two peaks of activity. Apparent purity of the major peak (TP I) was approximately 85-90%, as judged by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis. That of the minor fraction (TP II) was less. The major band of both fractions had a molecular mass of approximately 20,000 daltons. Both proteins catalyzed the transfer of ganglioside GM1 as well as asialo-GM1, but transfer protein I was more effective with di- and trisialogangliosides. Transfer protein II appeared to be somewhat more specific for neutral glycolipids in that GA1 was transferred more rapidly than any of the gangliosides; however, lactosylceramide transfer was relatively slow. Neither protein catalyzed transfer of phosphatidylcholine.     

Isolation and characterization of calcineurin from bovine brain

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid isolation of monosialogangliosides from bovine brain gangliosides by selective-overload chromatography.

A procedure for rapid isolation of monosialogangliosides from purified bovine brain gangliosides has been developed. It utilizes the selective difference in association between monosialogangliosides and polysialogangliosides for the ion-exchange resin Q-Sepharose. When the ion-exchange column is overloaded with a bovine brain ganglioside mixture in the proper ganglioside to column bed-volume ratio, the polysialogangliosides are selectively retained by the column while the monosialogangliosides emerge with the void volume without the use of salt for elution. With the critical ganglioside to bed-volume ratio (1 g:8.32 ml), and an appropriate column bed-height to column radius ratio of 6.9, monosialogangliosides are reproducibly obtained in high purity with greater than 90% yield. The method has been used at both the analytical and preparative scale. We call this separation technique selective-overload chromatography.     

Isolation and characterization of cathepsin B from bovine brain

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75–5.2 with the major band at pH 5.1–5.2, all of which were capable of degrading N-carbobenzoxy-l-arginyl-l-arginine 4-methoxy--naphthylamide. The cathepsin B activity against N-benzoyl-dl-arginine -naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.     

Phase behavior and structural characteristics of hydrated bovine brain gangliosides

Hydrated bovin brain gangliosides have been studied by differential scanning calorimetry, X-ray diffraction, and polarized light microscopy. Over the hydration range 18–50 wt.% H₂O, mixed brain gangliosides exhibit a hexagonal mesophase structure, in which the ganglioside molecules form hexagonally packed rod-like structures. The apolar lipid chains radiate from the center of the rods, with the sugar groups on the cylinder surface in contact with water. At higher water contents, an isotropic micellar solution is formed.Over the hydration range 20–30 wt.% H₂O, two small thermal transitions with peak maxima at 30°C and 46°C are observed by differential scanning calorimetry. These transitions broaden and move apart in temperature as the hydration is increased to 50 wt.% H₂O. X-ray diffraction data indicate that this double transition is associated with a hydrocarbon chain rearrangement from a disordered state to another, possibly more disordered, state. Thus, the gangliosides, although membrane lipid components, have physical characteristics which are very different from those of the membrane phospholipids.     

Isolation of three types of Gi from bovine spleen

We have previously reported the purification of two alpha subunits of G proteins, Gi2 and Gi3, from bovine spleen. However, it recently became clear that the preparation of Gi3 alpha contained a significant amount of Gi1 alpha by the immunoblot analysis using specific antibodies. In this study, we purified these G proteins as a trimer form from bovine spleen, and obtained following results. (1) Gi3 was separated from Gi1 using Mono Q column chromatography. Isoelectric focusing was employed to distinguish Gi3 from Gi1 in the column eluates. (2) Purified Gi2 and Gi3 retained much higher activities to bind GTP gamma S or to be ADP-ribosylated by pertussis toxin than the alpha subunits purified previously. (3) Using these spleen Gi2 and Gi3 and bovine brain Gi1, the parameter of GTP gamma S binding to the three types of Gi was compared. Three Gis showed different rates of GTP gamma S binding but showed the similar Kd values.     

Isolation and characterization of two novel peptide amides originating from myelin basic protein in bovine brain

During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-amide and Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-amide. These 12 and 16 residues peptides had the primary structure identical to the N-terminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and-16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain.     

Characterization of gangliosides from bovine erythrocyte membranes

Two glucosamine-containing gangliosides, sialosylhexaglycosylceramides, were isolated from bovine erythrocyte membranes. Both gangliosides were hydrolyzed by neuraminidase isolated from Clostridium perfringens to become neutral hexaglycosylceramides. Based on the results of sequential enzymatic hydrolysis and gas chromatography-mass spectrometric analyses of the methylated sugars, the structures of these two gangliosides were shown to be NeuAcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide and NeuGcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide, respectively. In addition, N-acetyl- and N-glycolylneuraminosyllacto-N-neotetraosylceramides, and N-acetyl- and N-glycolylneuraminosyllactosylceramides were also found in bovine erythrocytes. The predominant fatty acids in these two gangliosides were C 22:0 and C 24:0. C-18 sphingosine was the major base detected.     

Isolation of micro glutamic acid-rich protein from bovine brain

An acidic protein, designated as micro glutamic acid-rich protein, was purified to homogeneity from bovine brain extract, and was characterized in its physicochemical properties. The protein had an isoelectric point of 3.9, a molecular weight of 10,000, and was composed of very limited amino acid constituents; $\text{Asp}\text{Asn}$, Thr, Ser, $\text{Glu}\text{Gln}$, Pro, Gly, Ala and Lys, with a relative abundance of glutamic acid/glutamine which accounted for 51 % of the total amino acid composition. The yield of the protein was 750 μg/kg of wet brain tissue. The amino-terminal sequence analysis suggested that the protein arose through proteolysis of the 58,000-dalton precursor protein, that had been reported in a previous paper [Ishioka $\text{et al}$, (1980) Biochim. Biophys. Acta, 625, 281–290].     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Macrophage migration inhibitory factor: Isolation from bovine brain

The purification of macrophage migration inhibitory factor (MIF) from bovine brain cytosol and its partial characterization are reported. A rapid and relatively simple method for MIF isolation was developed based mainly on size-exclusion chromatography on Toyopearl TSK polymer having a tendency to adsorb MIF as compared to elution of other proteins with similar molecular weights. The method gives a high yield of MIF (0.1 mg homogenous protein per g wet tissue). The retardation is conveniently utilized to achieve good separations of MIF from other proteins of similar molecular weights. The isolated protein was identified as MIF by SDS-electrophoresis, immunoblotting, sequencing of the N-terminal amino acid residues, and also by determination of keto-enol tautomerase activity that is characteristic of MIF with p-hydroxyphenylpyruvic acid as a substrate.     

Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain

In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.     

Structures of gangliosides from bovine adrenal medulla.

Five gangliosides, accounting for over 95% of the total ganglioside fraction, were isolated from bovine adrenal medulla by preparative thin-layer chromatography and the carbohydrate structures determined by a combination of periodate oxidation and permethylation techniques. Partially methylated alditol acetates were generated from the neutral sugars of the fully methylated glycolipids and identified by gas-liquid chromatography. Substitution on N-acetylgalactosamine was determined by methanolysis of the permethylated ganglioside, acetylation of the products, and identification of the resulting substituted methyl glycosides by GLC. Periodate oxidation followed by borohydride reduction confirmed some of the linkages and demonstrated the absence of (2-8) linkages between sialic acid units. Mass spectrometry of the permethylated gangliosides gave information on sugar sequence at the nonreducing end.     

Isolation and characterization of major gangliosides from frog liver

Four major gangliosides isolated from frog liver were characterized by compositional analysis involving GLC and GC-MS, methylation analysis, chromium trioxide oxidation, and enzymatic hydrolysis. The results revealed that the most major ganglioside in the tissue was GM4 containing N-acetylneuraminic acid and the others were GM4 containing N-glycolylneuraminic acid, GD1a, and a fucosyl ganglioside which was tentatively assigned to be alpha-galactosyl alpha-fucosyl GM1. This is the first report describing the presence of GM4 containing N-glycolylneuraminic acid. The fatty acids in both GM4 were mainly alpha-hydroxylated, and those in the fucosyl ganglioside were exclusively nonhydroxy fatty acids. The GD1a contained both nonhydroxy and alpha-hydroxy fatty acids in a ratio of about 3:2. The predominant species were 22:0, 23:0, 24:0, and 24:1 in both species of the fatty acids. The long-chain bases of these four gangliosides consisted of C18-sphingosine and C18-phytosphingosine together with significant amounts of C16 to C19 dihydroxy and trihydroxy bases with iso and anteiso structures.