Dallas J. Hartman

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (1→3)-β-D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent Mr 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (1→3)-β-glucanases GI and GII have pI values of 8.6 and ≥ 10.0, respectively. Comparison of the sequences of their 40 NH2-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (1→3)-β-glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (1→3)-β-glucanase to be deduced, together with that of a putative NH2-terminal signal peptide of 28 amino acid residues. The (1→3)-β-glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (1→3)-β-glucanase shows highly conserved internal domains and 52% overall positional identity with barley (1→3, 1→4)-β-glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (1→3)-β-glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (1→3, 1→4)-β-glucanases, which function to depolymerize endosperm cell walls in the germinated grain.

Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES

Pig heart mitochondrial malate dehydrogenase was chemically denatured in guanidine HCl. Upon 50‐fold dilution of the denaturant spontaneous refolding could be observed in the temperature range 12–32°C. At 36°C spontaneous refolding was not observed but a stable folding intermediate that is fairly resistant to aggregation was formed. This intermediate is readily refolded by the chaperonins GroEL and GroES and may prove useful in future attempts to describe several aspects of chaperonin action at physiological temperatures.

The complete primary structure of rat chaperonin 10 reveals a putative βαβ nucleotide-binding domain with homology to p21ras

The first complete amino-acid sequence of a mitochondrial chaperonin 10 is reported. The amino-terminal alanine residue is acetylated, a modification that may be required for the interaction with heptameric chaperonin 60. Part of the sequence constitutes a potential dinucleotide binding motif and is identical with 7 out of 10 residues in the GTP-binding site of p21ras. This similarity may be the structural basis for the recently discovered complex between p21ras and chaperonin 60 in intact cells

Heat shock proteins of barley mitochondria and chloroplasts. Identification of organellar hsp 10 and 12: putative chaperonin 10 homologues.

Tissue slices from barley seedlings were subjected to heat shock and metabolically labelled with [35S]methionine and [35S]lcysteine, Mitochondria and chloroplasts were isolated and shown to contain two novel heat shock proteins of 10 and 12 kDa, respectively. The possibility that these proteins, like a mitochondrial 10 kDa stress protein recently isolated from rat hepatoma cells [(1992) Proc. Natl. Acad. Sci. 89, in press] represent eukaryotic chaperonin 10 homologues is discussed.

Identification of individual 1->3,1->4-β-D-glucanase isoenzymes in extracts of germinated barley using specific monoclonal antibodies.

Specific monoclonal antibodies have been used to distinguish between individual barley (1 → 3,1 → 4)-β-glucan endohydrolase (EC 3.2.1.73) isoenzymes-in enzyme linked immunosorbent (ELISA) and Western transfer assays. In addition, a monoclonal antibody that recognises both isoenzymes has been isolated. The antibodies, which are of the immunoglobulin IgG1 subclass and carry kappa light chains, do not cross-react with barley (1 → 3)-β-glucanases or with other proteins found in water-soluble extracts of germinated barley grain. The potential applications of these specific antibodies in barley breeding programmes and in physiological studies are discussed.