Cerebrosides hydrolysis

Fig. 2. High-performance liquid chromatographic analysis of biphenyl-carbonyl derivative of psychosine standards obtained by cerebroside hydrolysis, f represents point of injection. Abbreviation S.F.= sol-vent front.

CiaH3 7N02,CH3-[CH2],2-CH CH.CH(0H). CH(NH2>-CH20H. a base, forming part of the molecules of sphingomyelins and the cerebrosides, from which it splits off on hydrolysis. It crystallizes in needles from ether. 

Arylsulfatase [EC 3.1.6.1 ], also known simply as sulfatase, catalyzes the hydrolysis of a phenol sulfate, thereby producing a phenol and sulfate. This enzyme classification represents a collection of enzymes with rather similar specificities. (1) Steryl-sulfatase [EC3.1.6.2],also referred to as arylsulfatase C and steroid sulfatase, catalyzes the hydrolysis of 3-j8-hydroxyandrost-5-en-17-one 3-sulfate to 3-j8-hydroxyandrost-5-en-17-one and sulfate. The enzyme utilizes other steryl sulfates as substrates. (2) Cere-broside-sulfatase [EC 3.1.6.8], or arylsulfatase A, catalyzes the hydrolysis of a cerebroside 3-sulfate to yield a cerebroside and sulfate. The enzyme will also hydrolyze the galactose 3-sulfate bond present in a number of lipids. In addition, the enzyme will also hydrolyze ascorbate 2-sulfate and other phenol sulfates. 

This enzyme [EC 3.1.6.8], also known as arylsulfatase A, catalyzes the hydrolysis of a cerebroside 3-sulfate to produce a cerebroside and sulfate. The enzyme will also catalyze analogous reactions on the galactose 3-sulfate residues in a number of lipids as well as on ascorbate 2-sulfate and many phenol sulfates. 

A partial synthesis of cerebroside sulfate [ sulfatide, the glycoside of ceramide with galactose 3-sulfate, (90)] was achieved [90] by acylating the sphingosine galactosyl 3-sulfate (89) (obtained by basic hydrolysis of natural sulfatide) with palmitoyl chloride or D-2-acetoxypalmitoyl chloride (and subsequent basic hydrolysis of the... 

The mechanism of action of this activator has not been established, but it is known (Fischer and Jatzkewitz, 1978 Mitsuyama et al., 1985) that the activator forms an equimolar complex with cerebroside 3-sulfate prior to hydrolysis by arylsulfohydrolase A. Similar heat-stable activator proteins are also known for other acid hydrolases (Li and Li, 1983 Inui and Wenger, 1983 Wenger and Inui, 1984 Conzelmann et al., 1982 Christomanou and Kleinschmidt, 1985 Burg et al., 1985). It is becoming increasingly evident that many lysosomal hydrolases have specific activators, although some of these activators may be common to more than one enzyme (Li and Li, 1983, 1984 Li et al., 1985). The desulfation of cerebroside 3-sulfate also occurs in the absence of activator protein. In these experiments, Tween-20 or sodium... 

The diastereoisomeric configuration of the sugar-free cerebrosides (ceramides) was determined by Carter and coworkers,80 and found to be erythro, in accordance with the structure of sphingosine, which is isolated after drastic hydrolysis of sphingolipid mixtures or purified cerebrosides with acid. 

Cerebrosides have been isolated from Phycomyces blakeslearus (3), a fungus often found on animals dung, by extraction of mycelia with acetone and chloroform/methanol mixtures and purified on a silicic acid column, followed by a Florisil column. The bases obtained after hydrolysis were all phytosphingosine homologs ranging in length from C17 to C22. Palmitic,stearic, oleic, linoleic and hydroxy palmitic acids were the major fatty... 

Fluharty et aL (1974) have reported the isolation of S-labelled cerebroside sulphate. They injected the brains of young rats intracerebrally with [ S]-sulphate and sacrificed the animals 3 days later. Total lipids were extracted and glycerolipids destroyed by alkaline hydrolysis. TEAE-cellulose column chromatography was then used to purify the [ Sjsulphatide. Jatzkewitz and Nowoczek (1967) have synthesized o-galactose 3-sulphate and shown it to be identical to the galactose sulphate present in brain sulphatides. 

Belefonte, Pa, U.S.A. or obtained from brain cerebrosides by hydr lysis. Adult rat brain cerebroside was obtained by extraction with chloroform methanol 2 1. The extract was washed and passed through a silicic acid column and eluted with acetone methanol 9 1. Hydrolysis was carried out using the method described by Radin (1974) using butanol and aqueous KOH. 

Alkaline hydrolysis was carried out in order to remove PS and PE that can be extracted together with the psychosine. After the alkaline hydrolysis, the extract was washed three times with upper phase containing ammonium hydroxide. If the extract (checked by TLC) had still some contaminants as cerebrosides or sulphatides, a preparative TLC was carried out using chloroform methanol 2 N-NH3 60 35 8 as solvent in order to remove them. The spots were identified by spraying the plate with fluorescamine reagent in acetone (4-phenyl-spiro fu-ran-2 (3H) I -phtalan 3,3-dione) Hoffmann La Roche, Nut ley, U.S.A. 

Cerebrosides are soluble in hot ethanol, chloroform-methanol-mixtures insoluble in ether and cold ethanol. They can be purified by recrystallization from acetic acid or chloroform-methanol or by chromatography on activated magnesium silicate (Florisil) (Radin et al. 1959). Alkaline hydrolysis of the peptide bond with barium hydroxide (Klenk 1926) results in the formation of psychosine partial acid hydrolysis yields ceramide ... 

Hajra and Rad in (1965) have described a similar type of enzyme activity in pig brain preparations. The 100,000xg supernatant fraction catalyzes the hydrolysis of galactose containing cerebrosides. 

The index of hydrolysis was the formation of ste (-l- C)-sph from cerebrosides containing stearic-1- C acid. The use of a detergent mixture containing sodium cholate was required for hydrolysis. The enzyme showed maximum activity at pH 4.5, at which point both the enzyme and cholate precipitated. Other substrates included naturally occurring cerebrosides (galactose containing) and showed the formation of ceramides containing normal and a-hydroxy fatty acid. Enzyme activity was seen also in rat brain, spleen, and kidney. 

The importance of reliable methods is illustrated by the example of determining the concentration of cerebrosides in plasma. By measuring the increase in reducing power of a serum lipid extract before and after hydrolysis which was believed to be due to sugar released from cerebrosides Kirk (1938) found concentrations between 0 and 167 mg per 100 ml. With the use of purification procedures and specific reactions for sugars Svennerholm and Svennerholm (1958) were able to demonstrate a mean cerebroside content of 4.36 0.18 mg per 100 ml of plasma. 

With sphingolipids other than gangliosides, somewhat milder hydrolysis conditions were preferred, i.e. sphingomyelins were reacted for 1 hour in 1 M HCI in water-methanol (2 1 by volume) at 70°C for 16 hours, while cerebrosides required 3 M HCI in water-methanol (1 1 by volume) at 60 C for 1.5 hours [573]. (A related procedure, but for the isolation of the fatty acid constituents of sphingolipids for further analysis, has also been described [65] (see Chapter 4)). An objective experimental comparison of the above base- and acid-catalysed methods with a range of different substrates would now appear to be desirable. 

D-Galactose is obtained from the hydrolysis of lactose, its chief source from raffinose from the galactans of gums and mucilages and from the galactolipides, or cerebrosides, of the central nervous system. It resembles glucose, but is much less sweet, and less soluble in water. It is dextro-rotatoiy, [a] = + 80°. On partial oxidation, it forms the sparingly soluble mucic acid, by which it may be identified. 

Ceramide glucoside is the principal cerebroside present in peripheral organs, This material can be obtained chemic ly by controlled hydrolysis of more complex sphiiigoglycolipids, including the gai Iiosides (Svenner-holm, 1964 Ledeen, 1966). Such observations have resulted in this compound being assigned a central role in complex sphingt Iycoiipid metabolism,... 

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