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Role of laforin, a dual-specificity ...

Wang, Wei.

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Role of laforin, a dual-specificity protein phosphatase, in glycogen metabolism.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Role of laforin, a dual-specificity protein phosphatase, in glycogen metabolism./
作者:	Wang, Wei.
面頁冊數:	65 p.
附註:	Source: Dissertation Abstracts International, Volume: 67-02, Section: B, page: 0715.
Contained By:	Dissertation Abstracts International67-02B.
標題:	Biology, Molecular. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3203865
ISBN:	9780542526909

Role of laforin, a dual-specificity protein phosphatase, in glycogen metabolism.
Wang, Wei.

Role of laforin, a dual-specificity protein phosphatase, in glycogen metabolism. - 65 p.

Source: Dissertation Abstracts International, Volume: 67-02, Section: B, page: 0715.

Thesis (Ph.D.)--Indiana University, 2006.

Lafora disease, a progressive myoclonus epilepsy, is an autosomal recessive disease caused in about 45% of cases by mutation of the EPM2A gene, which encodes a dual specificity protein phosphatase called laforin. The disease is characterized by the appearance of Lafora bodies, deposits that contain polyglucosan or poorly branched glycogen. In addition to its phosphatase domain, laforin contains an N-terminal carbohydrate-binding domain (CBD). Mouse laforin was expressed as an N-terminally polyHis tagged protein in Escherichia coli and purified close to homogeneity. The enzyme was active towards para-nitrophenylphosphate (10-16 mumol/min/mg, Km 0.42 muM) with the maximal activity at pH 4.5. Glycogen binds to laforin through CBD and caused potent inhibition with half maximal effect at &sim;1 mug/ml, and less branched glucose polymers were even more potent. With all polysaccharides, however, inhibition was incomplete and laforin retained about 20% of its native activity at high polysaccharides concentrations. Glucose and short oligosaccharides did not affect activity. Substitution of Trp32 in the CBD by Gly, a mutation found in a patient, caused only a 30% decrease in laforin activity but abolished binding to and inhibition by glycogen, indicating that impaired glycogen binding is sufficient to cause Lafora disease. A genetic link between laforin and cellular glycogen content has been established by analyzing mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin expression while mice completely lacking muscle glycogen or with less muscle glycogen have reduced laforin. Mice defective in the gene coding lysozomal alpha-glycosidase overaccumulate glycogen in the lysosome and did not have elevated laforin suggesting that laforin senses cytosolic glycogen. Glycogen metabolizing enzymes were analyzed in a transgenic mouse strain overexpressing a dominant negative form of laforin. Lafora bodies accumulate in several tissues including muscle. The skeletal muscle glycogen was increased two-fold as was the total glycogen synthase protein. The -/+ glucose-6-phosphate activity of glycogen synthase, however, was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged as was glucose-6-phosphate concentration. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Glycogen metabolism in the skeletal muscle of this mouse model is modestly altered but does not provide an obvious explanation for the formation of Lafora bodies. All results in this thesis work gave evidence that laforin links to glycogen metabolism even though the underlying mechanism is still not clear.

ISBN: 9780542526909Subjects--Topical Terms:

1017719
Biology, Molecular.

Role of laforin, a dual-specificity protein phosphatase, in glycogen metabolism.
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520 $a Lafora disease, a progressive myoclonus epilepsy, is an autosomal recessive disease caused in about 45% of cases by mutation of the EPM2A gene, which encodes a dual specificity protein phosphatase called laforin. The disease is characterized by the appearance of Lafora bodies, deposits that contain polyglucosan or poorly branched glycogen. In addition to its phosphatase domain, laforin contains an N-terminal carbohydrate-binding domain (CBD). Mouse laforin was expressed as an N-terminally polyHis tagged protein in Escherichia coli and purified close to homogeneity. The enzyme was active towards para-nitrophenylphosphate (10-16 mumol/min/mg, Km 0.42 muM) with the maximal activity at pH 4.5. Glycogen binds to laforin through CBD and caused potent inhibition with half maximal effect at &sim;1 mug/ml, and less branched glucose polymers were even more potent. With all polysaccharides, however, inhibition was incomplete and laforin retained about 20% of its native activity at high polysaccharides concentrations. Glucose and short oligosaccharides did not affect activity. Substitution of Trp32 in the CBD by Gly, a mutation found in a patient, caused only a 30% decrease in laforin activity but abolished binding to and inhibition by glycogen, indicating that impaired glycogen binding is sufficient to cause Lafora disease. A genetic link between laforin and cellular glycogen content has been established by analyzing mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin expression while mice completely lacking muscle glycogen or with less muscle glycogen have reduced laforin. Mice defective in the gene coding lysozomal alpha-glycosidase overaccumulate glycogen in the lysosome and did not have elevated laforin suggesting that laforin senses cytosolic glycogen. Glycogen metabolizing enzymes were analyzed in a transgenic mouse strain overexpressing a dominant negative form of laforin. Lafora bodies accumulate in several tissues including muscle. The skeletal muscle glycogen was increased two-fold as was the total glycogen synthase protein. The -/+ glucose-6-phosphate activity of glycogen synthase, however, was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged as was glucose-6-phosphate concentration. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Glycogen metabolism in the skeletal muscle of this mouse model is modestly altered but does not provide an obvious explanation for the formation of Lafora bodies. All results in this thesis work gave evidence that laforin links to glycogen metabolism even though the underlying mechanism is still not clear.
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