Helga Stan-Lotter

Adenine nucleotide binding sites in normal and mutant adenosine triphosphatases of Escherichia coli

Abstract Three types of assays were used to characterize adenine nucleotide binding sites on the Ca2+, Mg2+-activated ATPase of normal Escherichia coli and its unc A 401 and unc D 412 mutants. ADP was bound mainly at a single site in normal and mutant ATPase. In the absence of divalent cations ATP was bound at a single high-affinity and three low-affinity sites in normal and unc D ATPases. The 2′,3′-dialdehyde (oADP) obtained by periodate oxidation of ADP reacted with both low- and high-affinity sites whereas oATP was bound primarily at a low-affinity site. Two types of adenine nucleotide binding sites, a high-affinity site reacting with ATP and ADP and a low-affinity site for ATP, were detected by the effects of these nucleotides on the fluorescence of the aurovertin D-ATPase complex. This high-affinity site(s) was present in normal and mutant ATPases. However, the fluorescence response at both high- and low-affinity sites was modified in the unc D ATPase as a consequence of the abnormal β subunit in this enzyme. Normal fluorescence responses were not induced by the binding of oADP or oATP to the ATPases. ATP was bound at a single site on isolated α subunits of the enzyme. Since this site was not detected in the unc A ATPase, it is unlikely to be the high-affinity site detected in the intact enzyme or the binding site for the endogenous tightly bound adenine nucleotides found in the purified ATPase. It is more probable that the site detected on the isolated α subunit from the normal enzyme is that which binds oADP since this site was absent in the unc A ATPase. Pretreatment of the normal ATPase with either N, N′-dicyclohexyl-carbodiimide (DCCD) or with 4-chloro-7-nitrobenzofurazan (NbfCl), reagents which inhibit ATPase activity by reacting with a β subunit, affected binding of oADP to α subunit(s) but had less effect with oATP. Inhibition of oADP binding could be due to conformational changes induced in the α subunit by the reaction of DCCD and NbfCl with a β subunit, or to steric reasons. If the latter hypothesis is correct, the active site of the ATPase would be at the interface between α and β subunits of the enzyme.

Affinity labeling of purified Ca2+,Mg2+-activated ATPase of Escherichia coli by the 2′,3′-dialdehydes of adenosine 5′-di- and triphosphates

Abstract The 2′,3′-dialdehydes of ADP and ATP (oADP and oATP), obtained by periodate oxidation of ADP and ATP, inhibited the hydrolytic activity of the purified Ca 2+ .Mg 2+ -activated ATPase of Escherichia coli . Nonspecific labeling of amino groups by these dialdehydes was corrected by carrying out the reactions in the presence of 15 m m ATP. Two types of modification of “ATP-protectable” binding sites by oATP could be detected. The binding of 2 mol “ATP-protectable” oATP/mol ATPase was without affect on ATPase activity and still occurred in the hydrolytically inactive ATPase of an unc A mutant. The binding of a further 3 mol “ATP-protectable” oATP/mol ATPase resulted in almost complete loss of ATPase activity although much of the loss occurred during the binding of the first additional molecule of oATP. This additional ATP-protectable oATP binding did not occur in the unc A mutant and so resembled both the inhibitory effect of oADP on the ATPase activity of normal strains and its lack of binding to the unc A ATPase ( P. D. Bragg and C. Hou, 1980 , Biochem. Biophys. Res. Commun. 95 , 952–957). The “ATP-protectable” binding sites for oADP and oATP were located on the α subunit of the ATPase. Binding of oADP or oATP did not result in release of the tightly bound ADP and ATP from the enzyme. We conclude that separate binding sites for oADP and oATP occur on the α subunits of the E. coli ATPase and that the former may be the active site(s) for ATP hydrolysis while the latter are involved in regulation of the ATPase complex.

Isolation of a fourth cysteinyl-containing peptide of the α-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence

The rapid determination of cysteinyl residues by Creightons method [(1980) Nature 284, 487‐489] led to the discovery of a discrepancy between protein and DNA sequence data in the α‐subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320‐328]. We have isolated a cysteinyl‐containing decapeptide from the α‐subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re‐sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene.

Subunit distribution of the sulfhydryl groups of the F1 adenosine triphosphatase of Salmonella typhimurium

The number of sulfhydryl groups in each subunit of the F1 adenosine triphosphatase of Salmonella typhimurium was measured by the method of T. E. Creighton [1980, Nature (London) 284, 487-489]. The alpha, beta, gamma, delta and epsilon subunits of this enzyme contained 4, 1, 2, 2, and 0 sulfhydryl groups per molecule of subunit, respectively.