Nucleotide sequence of the ATPase A- and B-subunits of the halophilic archaebacterium Haloferax volcanii and characterization of the enzyme.
Using PCR with degenerate oligonucleotides, we amplified two conserved regions of the plasma membrane ATPase A (alpha)- and B (beta)-subunit-encoding genes from Haloferax volcanii, a halophilic archaeon. The amplified fragments were cloned and sequenced, and then used as homologous probes to clone genomic restriction fragments, one of which contained the nearly complete atpA- and atpB-gene. To complete the latter one, we sequenced a region of an overlapping fragment using synthetic oligonucleotides as primers. The deduced amino-acid sequence showed a high degree of similarity with the A and B sequences of Halobacterium halobium-ATPase, and a relatively high degree with Daucus carota V-ATPase, but less similarity to F-ATPase alpha- and beta-subunits. Like in V-ATPases, the A-subunit is more related to the catalytic F-ATPase beta-subunit, whereas B corresponds to alpha. Cross-reaction of antibodies against CF1-alpha (synthetic peptide) with B and CF1-beta with A of Haloferax volcanii confirmed this finding. The ATPase of Haloferax volcanii is a halophilic enzyme, the amino-acid sequences contain about 20% negatively charged residues. This is discussed in terms of adaption to hypersaline conditions. The enzyme is specific for ATP, we determined KM values for ATP in presence of Mn2+ and Mg2+, respectively. Other nucleotides, including GTP and ADP are competitive inhibitors of ATP hydrolysis. Despite of the high degree of similarity to the Halobacterium enzyme, the ATPase showed no sensitivity to most of the tested inhibitors of ATP hydrolysis. NEM, a specific inhibitor for V-ATPases inhibited the enzyme only slightly. Bafilomycin, NBD-Cl and nitrate showed no effect. These results will be discussed in context with some specific differences in the primary structure of the Haloferax A-subunit.
Steinert
K
K
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Germany.
Kroth-Pancic
P G
PG
Bickel-Sandkötter
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001105:Archaea / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015183:Restriction Mapping; D016415:Sequence Alignment
D000596:Amino Acids; D006180:Proton-Translocating ATPases