Members of the 70-kD heat shock protein family have been found in all free-living organisms investigated and in major compartments of eukaryotic cells where they are essential to a wide range of functions, including protein folding and targeting. We have isolated a mitochondrial homolog (mtHSP70) from rat liver using ATP agarose affinity chromatography. Its identity was confirmed on the basis of immunological analysis and Ca(2+)-dependent autophosphorylation. Using protein sequence obtained from the amino termius and nine endo Lys-C peptide fragments, we have employed oligonucleotides to isolate a full-length cDNA clone. The open reading frame encodes a protein of 679 amino acids and calculated M(r) 73,913 daltons. The sequence has a high degree of identity with other members of the HSP70 family, including Escherichia coli DnaK (51%), Saccharomyces cerevisiae SSC1p (65%), the constitutive cytosolic HSP70 from rat, HSC70 (46%), and the rat endoplasmic reticulum isoform, BiP, (49%). The cDNA encodes a precursor protein with a 46-amino-acid signal peptide that is absent from the protein isolated from rat liver. The protein also shows a high degree of identity (98%) with a protein isolated from mouse and human tissues (PBP74, Domanico et al., 1993; mortalin, Wadhwa et al., 1993a; CSA, Michikawa et al., 1993a); however, the intracellular localization of these proteins is uncertain. We show that the precursor of mtHSP70 is efficiently imported into isolated mitochondria from rat liver and processed from 74 kD to the mature 69-kD protein.

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 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 (Ikawa, S. and Weinberg, R.A. (1992) Proc. Natl. Acad. Sci. USA 89, 2012-2016).

The molecular chaperones GroEL and GroES were produced at very high levels in Escherichia coli, purified, and shown to protect pig mitochondrial malate dehydrogenase (MDH) against thermal inactivation in vitro. The apparent rate of MDH inactivation at 37 degrees C was reduced by a factor of at least 5 in a process which required only GroEL, GroES, and ATP. GroEL alone did not protect MDH against thermal inactivation but kept the denatured protein soluble and thereby prevented its aggregation. Reactivation of this soluble and inactive form of MDH could be achieved by addition of GroES even after 120 days of storage at -20 degrees C. Protection could be extended for more than 24 hr at 37 degrees C and was observed at molar ratios of chaperones to MDH as low as 1:4, suggesting that GroEL and GroES perform multiple turnovers in the absence of auxiliary chaperones. The availability of these chaperones in large quantities combined with the apparent promiscuity of GroEL binding shows great potential for stabilization of many proteins for which thermostable variants are not available. We speculate that GroEL and GroES perform similar protective roles in vivo and thereby increase the half-life of proteins which otherwise might aggregate under physiological conditions.

We have identified a 10-kDa stress-inducible mitochondrial protein. The protein is synthesized at elevated rates in cultured rat hepatoma cells challenged with heat shock or amino acid analogues and, therefore, designated heat shock protein 10 (Hsp10). Hsp10 was purified to homogeneity from rat liver and found to exhibit a native molecular mass of 65 kDa, as opposed to a monomeric molecular mass of 10,813.4 +/- 0.41 Da. The amino acid sequence of rat Hsp10 disclosed extensive sequence similarity with bacterial chaperonin (Cpn) 10. Rat Hsp10 and Escherichia coli Cpn60 were used to reconstitute functional trimeric rat ornithine transcarbamoylase from a chemically denatured state with high efficiency. This process depended completely upon rat Hsp10 and was abolished in the presence of a nonhydrolyzable ATP analogue. We conclude that Hsp10 is a eukaryotic Cpn10 homologue and, therefore, together with Cpn60 essential for mitochondrial protein biogenesis. The Cpn-mediated protein-folding apparatus, thus, exhibits a high degree of conservation between prokaryotes and mitochondria of higher eukaryotes.

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.

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.

A (1-->3)-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) of apparent M(r) 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)-beta-glucanases GI and GII have pI values of 8.6 and > or = 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)-beta-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)-beta-glucanase to be deduced, together with that of a putative NH2-terminal signal peptide of 28 amino acid residues. The (1-->3)-beta-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)-beta-glucanase shows highly conserved internal domains and 52% overall positional identity with barley (1-->3, 1-->4)-beta-glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (1-->3)-beta-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)-beta-glucanases, which function to depolymerize endosperm cell walls in the germinated grain.