Purification and characterization of (1-->3, 1-->4)-beta-glucan endohydrolases from germinated wheat (Triticum aestivum).
A (1-->3, 1-->4)-beta-glucan 4-glucanohydrolase [(1-->3, 1-->4)-beta-glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (1-->3, 1-->4)-beta-glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (1-->3, 1-->4)-beta-glucanase isoenzyme EI from barley. The complete primary structure of the wheat (1-->3, 1-->4)-beta-glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)+ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated lambda LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH2-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32,085 and a predicted pI of 8.1. The other cDNA, designated lambda LW1, carries a 109 nucleotide pair sequence at its 5' end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3'-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.
Lai
D M
DM
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Høj
P B
PB
Fincher
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002855:Chromatography, Thin Layer; D003062:Codon; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics / Q000502:physiology
D003062:Codon; D010940:Plant Proteins; D004247:DNA; D006026:Glycoside Hydrolases; C028020:licheninase