(1,3) beta-Glucan synthase activity of Neurospora crassa: identification of a substrate-binding protein.
(1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neurospora crassa was purified 1300-fold to a specific activity of 14,000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. Membrane fractions were extracted with Tergitol NP-40 and a second high-speed particulate fraction was obtained. Enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-beta-D-glucoside from Tergitol-extracted membrane preparations. Solubilized enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysis revealed 2 proteins of 165 and 100 kDa as likely candidates for subunits of the (1,3)beta-glucan synthase enzyme complex. 5-Azido-[32P]UDP-glucose was photo-crosslinked to UDP-glucose-binding proteins in each fraction of the purification procedure. Autoradiograms of SDS-PAGE gels revealed a single protein of 165 kDa enriching with enzyme activity and labeling with the substrate analog. Photoincorporation of 5-azido-[32P]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP-glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-glucose (36%), and GDP-glucose (8%) where less effective. These results were similar to in vitro inhibition of enzyme activity by the same compounds. These data strongly suggest that the 165 kDa protein is a substrate-binding subunit of (1,3)beta-glucan synthase.
Awald
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Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver 80262.
Frost
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Drake
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Selitrennikoff
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D002352:Carrier Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification*; D008565:Membrane Proteins; D009492:Neurospora crassa / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase