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Purification and characterization of...

Mu, He.

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Purification and characterization of starch phosphorylase from maize endosperm.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Purification and characterization of starch phosphorylase from maize endosperm./
作者:	Mu, He.
面頁冊數:	87 p.
附註:	Directors: George M. Carman; Bruce P. Wasserman.
Contained By:	Dissertation Abstracts International61-10B.
標題:	Agriculture, Food Science and Technology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9991913
ISBN:	0599995882

Purification and characterization of starch phosphorylase from maize endosperm.
Mu, He.

Purification and characterization of starch phosphorylase from maize endosperm. - 87 p.

Directors: George M. Carman; Bruce P. Wasserman.

Thesis (Ph.D.)--Rutgers The State University of New Jersey - New Brunswick, 2000.

A plastidic 112-kD starch phosphorylase has been identified in the amyloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymological and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch adsorption, Q-Sepharose chromatography, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-polyacrylamide gel electrophoresis. Anti-starch phosphorylase antibodies recognized the purified 112-kD starch phosphorylase enzyme and N-terminal amino acid sequence analysis confirmed that the purified enzyme is the amyloplast stromal 112-kD starch phosphorylase. Analysis of the purified enzyme by Superose 6 gel filtration chromatography indicated that the native enzyme consisted of two identical subunits. The pH optimum for the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolytic direction. Starch phosphorylase activity was inhibited by thioreactive agents, diethyl pyrocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 kcal/mol and 16.9 kcal/mol, respectively, and the enzyme was thermally labile above 50°C. Results of kinetic experiments indicated that the enzyme catalyzes its reaction via a sequential Bi Bi mechanism. The <italic>Km</italic> value for amylopectin was 8-fold lower than that of glycogen. A kinetic analysis indicated that the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (4 to 7 glucose units) were used as substrates. The specificity constants (<italic>Vmax</italic>/<italic>Km</italic>) of the enzyme measured in either the synthetic or the phosphorolytic directions increased with increasing chain length.

ISBN: 0599995882Subjects--Topical Terms:

1017813
Agriculture, Food Science and Technology.

Purification and characterization of starch phosphorylase from maize endosperm.
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245 1 0 $a Purification and characterization of starch phosphorylase from maize endosperm.
300 $a 87 p.
500 $a Directors: George M. Carman; Bruce P. Wasserman.
500 $a Source: Dissertation Abstracts International, Volume: 61-10, Section: B, page: 5072.
502 $a Thesis (Ph.D.)--Rutgers The State University of New Jersey - New Brunswick, 2000.
520 $a A plastidic 112-kD starch phosphorylase has been identified in the amyloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymological and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch adsorption, Q-Sepharose chromatography, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-polyacrylamide gel electrophoresis. Anti-starch phosphorylase antibodies recognized the purified 112-kD starch phosphorylase enzyme and N-terminal amino acid sequence analysis confirmed that the purified enzyme is the amyloplast stromal 112-kD starch phosphorylase. Analysis of the purified enzyme by Superose 6 gel filtration chromatography indicated that the native enzyme consisted of two identical subunits. The pH optimum for the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolytic direction. Starch phosphorylase activity was inhibited by thioreactive agents, diethyl pyrocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 kcal/mol and 16.9 kcal/mol, respectively, and the enzyme was thermally labile above 50°C. Results of kinetic experiments indicated that the enzyme catalyzes its reaction via a sequential Bi Bi mechanism. The <italic>Km</italic> value for amylopectin was 8-fold lower than that of glycogen. A kinetic analysis indicated that the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (4 to 7 glucose units) were used as substrates. The specificity constants (<italic>Vmax</italic>/<italic>Km</italic>) of the enzyme measured in either the synthetic or the phosphorolytic directions increased with increasing chain length.
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