The Recombinant Thermosome from the Hyperthermophilic Archaeon Methanopyrus kandleri: In Vitro Analysis of Its Chaperone Activity
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T. Minuth
Abstract
The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function.
In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coli BL21(DE3) cells. Purification yielded soluble, high-molecularmass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast α-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates.
The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 °C.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Artikel in diesem Heft
- To Our Authors, Readers and Subscribers. Biological Chemistry 1999 - Increased Page Number and Online Accessibility
- Proto-Oncogenes, Unlike Harmless Genes, Tend to Be Dispersed in the Human Genome: Selection Against Out-of-Register Recombination?
- The Human H2A and H2B Histone Gene Complement
- Efficient Control of raf Gene Expression by CAP and Two Raf Repressors that Bend DNA in Opposite Directions
- Molecular Models of Acidic PePtides From Pea Bud Chromatin And Seminal Plasma. Divalent Cations-Mediated Interaction With Dna
- Cytidine Triphosphate Synthase Activity and mRNA Expression in Normal Human Blood Cells
- Studies with Lysine N6-Hydroxylase. Effect of a Mutation in the Assumed FAD Binding Site on Coenzyme Affinities and on Lysine Hydroxylating Activity
- The Recombinant Thermosome from the Hyperthermophilic Archaeon Methanopyrus kandleri: In Vitro Analysis of Its Chaperone Activity
- Not More than Three Tissue Kallikreins Identified from Organs of the Guinea Pig
- Activation of Protein C by Arginine-Specific Cysteine Proteinases (Gingipains-R) from Porphyromonas gingivalis
- Alteration of Mosaic Methylation of the Repeat Unit of the Human Ribosomal RNA Genes in Lung Cancer
- Degradation of the Cyclic AMP Antagonist Prostaglandylinositol Cyclic Phosphate (Cyclic PIP) by Dephosphorylation
- Homo-Dimeric Spherulin 3a: A Single-Domain Member of the bg-Crystallin Superfamily
- cDNA Sequencing of Guinea Pig a2-HS Glycoprotein, Its Expression in Various Tissues and Acute Phase Expression
- Phage Display Selection of P1 Mutants of BPTI Directed against Five Different Serine Proteinases
