Takayoshi Wakagi

A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilic HB8: purification and general properties

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermus thermophilus HB8, and shown to be a dimeric protein of molecular weight 115,000, with a pI of 5.5. The amino acid composition of the present enzyme was similar to that reported for other bacterial counterparts, except for a high Arg/Lys ratio and a low Cys content. Divalent cations, such as Mn2+ and Mg2+, were essential for activity. The optimal pH was 7.8 at 55 degrees C. The Km values for NADP and D-isocitrate were 6.3 and 8.8 microM, respectively, with a Vmax of 77.6 mumol/min per mg at 55 degrees C. NAD was able to replace NADP with low efficiency. Backward reaction at 40 degrees C indicated that the Km value for 2-oxoglutarate was 63 microM with a Vmax of 4% that of the forward reaction at that temperature. The enzyme was highly stable against high temperature and denaturing reagents.

An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7

A highly active inorganic pyrophosphatase was purified to electrophoretical homogeneity from the cytosol of Sulfolobus acidocaldarius strain 7, an extremely thermoacidophilic archaebacterium. The enzyme has an apparent molecular mass of 80 kDa as estimated by gel permeation chromatography, and showed a 21-kDa polypeptide on SDS-PAGE, suggesting that the archaebacterial enzyme is similar to most of the eubacterial pyrophosphatases rather than eukaryotic ones. The pI = 5.1. The enzyme showed relatively high content of Pro and low content of Ser plus Thr. The optimal pH was 6.5 (at 56 degrees C). From the Arrhenius plot an activation energy of 11.2 kcal/mol was obtained between 37-95 degrees C. The specific activity was 617 mumol Pi release min-1 mg-1 at 56 degrees C. The S. acidocaldarius pyrophosphatase was extremely stable. Complete activity remained after incubation at 100 degrees C for 10 min. No dissociation into subunit or unfolding of polypeptide chain occurred in the presence of 8 M urea. Experiments using guanidine-HCl suggested that the transition between a native tetrameric state and an unfolded state is completely reversible, and essentially independent of any additional factors such as divalent metal cation or dithiothreitol.

Energy metabolism of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of the archaebacterium and mechanisms of acidophily, membrane bound ATPase, cytochromes and NADH dehydrogenase of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Typea cytochrome was found in the membrane. The organism was sensitive to cyanide and azide, and though cytochromec is lacking in this organism, these respiratory poisons inhibited a terminal oxidase, when assayed with cytochromec from other sources. NADH dehydrogenase was highly purified from the crude extract of the cells. The enzyme was able to transfer electrons from NADH to caldariellaquinone, a unique benzothiophenequinone in the genusSulfolobus. Thus, the enzyme is a possible member of the respiratory chain. Membrane fraction contained two types of ATPase, one was active at neutral pH and slightly activated by sulfate; the other was an acid apyrase and inhibited by sulfate. Typical characteristics of F0F1ATPase could not be found in these enzymes. These results suggest that (1) the thermoacidophilic archaebacteria are phylogenically distant from both eubacteria and eukaryotes, (2) the archaebacterial thermoacidophiles can be classified in a different subgroup from methanogens and extreme halophiles, and (3) in spite of the aerobic nature of the organism, the energy yielding mechanisms appear quite unique, when compared to those of other aerobes and mitochondria.