Bovine kidney mitochondria were separated into matrix and membrane fractions by treatment with digitonin and Lubroi PX. While maiate dehydrogenase was found essentially in the matrix fraction, both the pyruvate and the a-oxoglutarate dehydrogenase multienzyme complexes remained bound to the inner membrane fraction and became solubilized only after repeated treatments with detergents. Thus both multienzyme complexes must be associated with the inner membrane rather than located within the matrix space.

Colicin B was isolated in pure form from Escherichia coli Cl139 and was shown to consist of a single polypeptide chain with an apparent Mr of 70000. Therefore, it does not differ from other colicins where the toxic activity resides in one polypeptide.

The pyruvate dehydrogenase complex from the thermophilic bacterium Thermus aquuticus was purified by Triton X-100 extraction and chromatography on phenyl-Sepharose CL-4B and HPLC-hydroxyapatite. The electrophoretic pattern of the purified enzyme complex was similar to that of the enzyme complex from Bacillus subtifis, with four bands: the a-chain (Mr 39600) and @-chain (Mr 37500) of the pyruvate dehydrogenase component, the dihydrolipoamide acetyltransferase component ( Mr 58500) and the dihydrolipoamide dehydrogenase component (Mr 53900). Antibodies against the purified T. aquuticus pyruvate dehydrogenase complex cross-reacted with the enzyme complex from B. subtilis and, to a minor extentiwith that from bovine heart. No cross-reactivity could be observed with the enzyme complex from Escherkhiu coli. The T. uquuticus enzyme complex had a temperature maximum at 72 OC. 2-Oxobutyrate was a poor substrate and other 2-oxoacids were competitive inhibitors of the overall reaction. Long-chain 2-oxoacids showed a greater inhibitory effect, possibly caused by hydrophobic interactions. GTP inhibited the enzyme activity. Regulation of the pyruvate dehydrogenase complex from T. aquaticus by allosteric mechanisms or by reversible phosphorylation could not be demonstrated.

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