Purification, characterization and gene structure of (1-->3)-beta-glucanase isoenzyme GIII from barley (Hordeum vulgare).
A new member of the barley (1-->3)-beta-glucan glucanohydrolase family of enzymes has been purified from extracts of germinated grain and young seedlings by fractional precipitation with ammonium sulphate, ion-exchange chromatography, chromatofocussing and gel-filtration chromatography. The enzyme, which has been designated (1-->3)-beta-glucanase isoenzyme GIII, is a basic protein with an apparent molecular mass of 32 000 Da. Oligosaccharide products released by the enzyme during hydrolysis of the (1-->3)-beta-glucan, laminarin, indicate that the enzyme is an endohydrolase. A 2349-bp fragment of barley genomic DNA has been isolated and identified as the gene encoding the (1-->3)-beta-glucanase isoenzyme GIII. The open reading frame encoding the isoenzyme is interrupted by a single intron of 180 bp that splits a codon in the putative signal-peptide region. Northern-blot analyses with gene-specific probes indicate that the (1-->3)-beta-glucanase isoenzyme GIII mRNA accumulates in developing leaves; no mRNA transcripts were detected in the aleurone or scutellum of germinated grain, or in mature vegetative tissues. Although plant (1-->3)-beta-glucanases are generally classified as 'pathogenesis-related' proteins, the physiological function of the barley (1-->3)-beta-glucanase isoenzyme GIII is unclear.
Wang
J
J
Department of Biochemistry, La Trobe University, Victoria, Australia.
Xu
P
P
Fincher
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D017343:Genes, Plant; D043326:Glucan 1,3-beta-Glucosidase; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase