Molecular evolution of plant beta-glucan endohydrolases

Peter B. Høj and Geoffrey B. Finche

Charaterisation of several Hsp70 interacting proteins from mammalian organelles

Since both the spectrum and characteristics of in vivo substrates with affinity for Hsp70 members are largely unknown, we have investigated the range and type of mammalian organellar proteins which selectively interact with immobilised Escherichia coli Hsp70 (DnaK). Amongst a subset of organellar proteins selectively retained on DnaK, the major constituents represent unstable proteins and subunits of oligomeric proteins. The interactions with DnaK were diminished in the presence of mt-Hsp70 and BiP, while the complexes formed with DnaK were dissociated in the presence of K+ and GrpE-like co-chaperones, suggesting that these organellar proteins constitute general Hsp70 substrates. Protein sequence analysis identified the major DnaK interacting constituents as the mitochondrial transcription factor A, the α- (but not the β-) subunit of succinyl CoA synthetase, mitochondrial 2,4-dienoyl CoA reductase, endoplasmic reticulum cyclophilin-B, peroxisomal multifunctional enzyme and a previously undescribed peroxisomal protein suspected to represent an isoform of 2,4-dienoyl CoA reductase. The selective retention of these fully synthesised proteins on Hsp70 most likely reflects the function of this molecular chaperone in protein biogenesis, but additionally, could extend the known functions of Hsp70 to include modulating the activities of certain proteins or enzymes which are important in cellular homeostasis

Purification and characterization of ornithine acetyltransferase from Saccharomyces cerevisiae

Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme catalyses the freely reversible interchange of an acetyl group between N-acetylornithine and glutamate and has a specific activity of 22 μmol · min−1· mg−1 at 37°C and pH 7.5. The Km values were determined for the substrates of the forward and reverse directions to be 1.0 mM for N-acetylornithine, 7.2 mM for glutamate, 1.5 mM for ornithine and 17.1 mM for N-acetylglutamate. The enzyme was localised to the mitochondrial matrix and was found to be a 57-kDa heterodimer consisting of subunits of 31 kDa and 26 kDa. Antibodies raised against the small subunit immunoprecipitated a single in vitro translation product of approximately 57 kDa suggesting that the subunits are processed from a single precursor protein. This is supported by N-terminal sequence analysis which shows that the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) with the N-terminus of ornithine acetyltransferase from Neisseria gonorrhoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identity (9 out of 20) with a sequence located in the middle of the 60-kDa N. gonorrhoeae enzyme. The N-terminal sequence of the small subunit has the potential to form an amphiphilic helix, further suggesting that the precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits