Cyclitol containing carbohydrates from human tissue which regulate glycogen metabolism
AssigneeRademacher Group Limited
InventorsThomas William Rademacher, Hugo Caro
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Abstract
The present invention provides for an isolated P-type substance having a structure identical to a P-type substance obtained from human liver or placenta, which is a cyclitol containing carbohydrate comprising Mn2+ and Zn2+ and related compositions.
Claims23
An isolated P-type substance as obtainable from human liver or placenta, wherein the substance is a cyclitol containing carbohydrate comprising Mn2+ and Zn2+ ions and has the biological activity of activating pyruvate dehydrogenase (PDH) phosphatase.
An isolated P-type substance which is a cyclitol containing carbohydrate, said substance comprising Mn2+ and Zn2+ ions, as obtainable from human liver or placenta by:
- (a) making an extract by heat and acid treatment of a liver or placenta homogenate, the homogenate being processed from tissue immediately frozen in liquid nitrogen;
- (b) centrifuging the extract to produce a first supernatant, treating the first supernatant with charcoal, recentrifuging the treated first supernatant to produce a second supernatant, and treating the second supernatant overnight with an AG1-X8 anion exchange resin to produce a second supernatant/resin solution;
- (c) pouring the second supernatant/resin solution into a column and eluting the P-substance from the column with 10 mM HCl to produce an acid eluate containing the P-type substance;
- (d) neutralising the acid eluate to between pH 4 and pH 7.8 and lyophilising the neutralized acid eluate to produce a first preparation containing the P-type substance;
- (e) subjecting the first preparation to descending paper chromatography using 4/1/1 butanol/ethanol/water as solvent to obtain a second preparation containing the P-type substance;
- (f) subjecting the second preparation to high voltage paper electrophoresis in pyridine/acetic acid/water to obtain a third preparation containing the P-type substance;
- and,
- (g) further purifying the P-type substance by subjecting the third preparation to Dionex anion exchange chromatography and/or isolating the P-type substance using Vydac HPLC chromatography.
An isolated substance which is a P-type cyclitol containing carbohydrate comprising Mn2+ and Zn2+ ions and has the biological activity of activating pyruvate dehydrogenase (PDH) phosphatase, wherein the substance has a molecular weight determined using negative mode MALDI mass spectroscopy as shown in FIG. 11, or a molecular weight related to one of the molecular weights set out in FIG. 11 by the addition or subtraction of one or more structure units of about 236 m/z.
Dependencies are inferred from the claim text.