Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons.
A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.
Kamat
U
U
Department of Biosciences and Biotechnology, University of Roorkee, India.
Garg
R
R
Sharma
C B
CB
D010367:Arachis / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D017886:Durapatite; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D006882:Hydroxyapatites; D007700:Kinetics; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D012265:Ribonucleotides / Q000494:pharmacology; D029702:Schizosaccharomyces pombe Proteins; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity
D003902:Detergents; D006882:Hydroxyapatites; D008565:Membrane Proteins; D012265:Ribonucleotides; D029702:Schizosaccharomyces pombe Proteins; D017886:Durapatite; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase