Cerebroside oxidize

We obtained unexpected findings using this method to study myelin the oxidation by galactose oxidase of myelin-bound cerebrosides could not be detected. The oxidation did not occur either with the intact spinal cord preparation, with isolated myelin, or even with lyophilized myelin. In one experiment, lyophilized myelin containing 5 mg each of dry weight was incubated with 100, 200, and 500 units of galactose oxidase for 60 min at room temperature, and no cerebroside oxidation occurred. 

Treatment with galactose oxidase. Oxidation of myelin with galactose oxidase was performed as described previously for similar oxidation of rat spinal cord preparations (4). Typically, myelin containing 0.2-1.1 mg protein is incubated with 100-500 units of galactose oxidase in 1-3 ml of phosphate buffer (10-100 mM, pH 7.2-7.4) with or without catalase. After the incubation at room temperature to 30°C for the duration of 30 min to overnight, myelin is recovered by centrifugation, washed, and lyophilized. Total lipids were extracted from the dried residue and the oxidized cerebroside as well as unaltered cerebrosides were analyzed as described above. Alternatively, the incubation was stopped by the addition of 5 volumes of chloroform/methanol (2/1, v/v) and mixed. The lower layer after centrifugation of the mixture is washed and then evaporated to dryness, and the total lipids obtained were analyzed as described above. 

Oxidation of myelin surface cerebrosides by galactose oxidase. Fig. 4 shows silica HPLC of a mixture containing benzoylated-non-hydroxy and hydroxycerebroside and benzoylated derivatives of 2,4-dinitrophenylhydrazone of oxidation products from nonhydroxy- and hydroxycerebroside. Standard curves of two 6-dehydro-derivatives were shown in Fig. 5. These standard curves demonstrate that the response of the benzoylated dinitrophenylhydrazones are linear between 0.025 nmol and 0.6 nmol. Since cerebrosides containing 5 nmol can be determined without tailing to these peaks, this method should allow the determination of as little as 0.5% of the oxidation product. The fact that each curve intersects 0 point in both the abscissa and ordinate indicates that even smaller amounts of these compounds can be detected by this technique. 

To examine whether the enzyme is active under the same conditions, we coated 0.1 mg each of nonhydroxy- and hydroxycerebrosides on 10 mg Celite (Analytical grade) and incubated it with 100 units of galactose oxidase for 60 min at room temperature. The result indicated that 5.6 nmol and 3.5 nmol each of nonhydroxy and hydroxycerebrosides (approximately 4.6 and 3.0% each were oxidized. Oxidation of the same cerebrosides by the same galactose oxidase in a tetrahydrofuran/water mixture as described by Radin (9) resulted in nearly complete oxidation. 

To further examine the inability of galactose oxidase in oxidizing myelin-bound cerebrosides, one mg each of lyophilized... 

Oxidation of other forms of cerebrosides by galactose oxidasel Microsomes (5.49 mg protein) and cytosol (7.15 mg protein) from 25 day-old rat brain, which were prepared as described previously (15), were each incubated with 100 units of galactose oxidase overnight at room temperature. The results indicate that oxidation of cerebrosides in these brain sub-cellular fractions was not detected. We tested whether cerebrosides in artificial membrane could be oxidized by this enzyme. Liposomes were prepared as described in Procedures. 0.2 ml Of the liposomes which contained 40 yg nonhydroxycerebroside and 33 yg hydroxycerebroside was incubated with 100 units of galactose oxidase at room temperature overnight. The examination of the product as described above shows that cerebrosides in liposomes was not oxidized. Another aliquot (0.2 ml) of the liposomes was mixed with 1 ml tetrahydrofuran, 1.0 ml of 10 mM phosphate buffer, pH 7.2 containing 100 units of galactose oxidase. More than 90% of cerebrosides were oxidized by this method. 

The second cause may be due to steric hinderance of neighboring components within the myelin sheath. However, we found that digestion of trypsin or phospholipase C cannot alleviate the problem. In addition, hypotonic treatment of myelin which affects the integrity of the bilayer and also causes the splitting of the lamellae at the external apposition (19, 20), did not result in the oxidation of the cerebrosides. Even cerebrosides in liposomes made from pure phosphatidyl choline could not be oxidized. Incidentally, this finding also contradicts Linington and Rumsby who reported significant oxidation of cerebrosides in liposomes made from myelin lipids. 

From saponification of cerebrosides, lipids found in the membranes of brain and nerve cells therc is obtained nervonic acid. This acid rapidly decolorizes dilute KMn04 and Br2/CCl4 solutions. Hydrogenation in the presence of nickel yields tetracosanoic acid, W-C23H47COOH. Vigorous oxidation of nervonic acid yields one acid of neutralization equivalent 156 3 and another acid of neutralization equivalent 137 2. What structure or structures are possible for nervonic acid ... 

Both the reduced periodate-oxidized cerebroside and the ceramide were as active as the original cerebroside, suggesting that the sugar moiety is not important for activity. The sphingoid moiety of a cerebroside constitute an essential part of the activity and the fatty acid having a certain chain length and the 2-hydroxy fatty acids may be the functional structures of this glycolipid (80). 

Hydroxy FA occnr in both satnrated and nnsatnrated forms, with varions positional isomers. There are three major types. First, the 2-hydroxy (2-OH) or a-hydroxy FA (Fignre 3.1). These satnrated components occnr as a series of even nnmbered acids from Cm to C26 and are components of animal tissnes (within cerebrosides) and certain plants. Second, 3-hydroxy (3-OH) or P-hydroxy FA exist as a series of even-nnmbered acids from Cm to C. They are ubiquitous components of many bacteria and yeasts, where they exist as ester-linked residues of extracellular lipids. Third, the hydroxyl group may be present at the penultimate carbon from the carboxyl group (i.e., co2, or otherwise referred to as co-1) of the chain series Cn to Cm- In most instances, these compounds represent intermediates in the co-oxidation of FA by microorganisms. 

Sulphation of lactose with pyridine-SOa complex in pyridine resulted in formation of the galactose 6-sulphate. Other oligosaccharides were alsostudied. I he glycolipids, seminolipid and cerebroside sulphate, which are sulphated on the 3-position of galactose, were not oxidized by galactose oxidase. 

Wang JW, Zheng LP, Zhang B, Zou T (2009) StimulatirHi of artemisinin synthesis by combined cerebrosides and nitric oxide elicitation in Artemisia annua hairy roots. Appl Microbiol Biotechnol 85 285-292... 

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