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.