The use of differing nucleotides to investigate cross-bridge kinetics.

We have investigated the ability of the nucleotides GTP, CTP, and 1-N6-etheno-2-aza-ATP (aza-ATP) to support contraction of chemically skinned rabbit psoas fibers. Working at 10 degrees C, millimolar concentrations of all nucleotides relaxed fibers in the absence of calcium. In active fibers, GTP served as a very poor substrate with isometric tension, isometric GTPase rate, and maximum shortening velocity (Vmax) all less than 10% of those obtained with ATP. Aza-ATP was only a slightly better substrate. CTP, on the other hand, was an effective substrate with mechanical parameters which were 65-100% those obtained with ATP, and with a hydrolysis rate that exceeded that of ATP. For all three ligands, Vmax followed Michaelis-Menten saturation behavior with values for Km which were from 2.5 to 12 times greater than that for ATP, showing that the analogs bound slowly to myosin in the fibers. Increasing concentrations of orthophosphate inhibited tension with CTP, to a lesser extent with aza-ATP, but not all with GTP. A combination of the mechanical data obtained in fibers with the kinetic data obtained in solution (White, H.D., Belknap, B., and Jiang, W. (1993) J. Biol. Chem. 268, 10039-10045) is used to better define the actomyosin interaction in fibers.

Disparate cytogenetic responses of peripheral blood and spleen lymphocytes to ethenoadenine nucleotides in vitro; maximal expression in splenic lymphocytes under conditions of enhanced membrane permeabilization.

Exogenously supplied 1,N6-ethenoadenosine triphosphate (epsilon-ATP) and 1,N6-ethenodeoxyadenosine triphosphate (epsilon-dATP) are potent inducers of sister chromatid exchanges (SCEs) in murine spleen lymphocytes but not in peripheral blood lymphocytes cultured in vitro. Data suggest that spleen lymphocyte membranes are inherently more permeable than blood lymphocytes to transient uptake of epsilon-ATP and epsilon-dATP. The effect of media pH and divalent cations on SCE frequency and chromosomal aberrations in spleen cells pulse-treated with epsilon-ATP were studied. The most dramatic responses were observed at pH 8.0 in Ca2+/Mg2(+)-free Hank's balanced salt solution (HBSS). Under the latter conditions, SCE and chromosomal aberration responses (mean +/- SD) of lymphocytes from replicate mice were 69.4 +/- 13.1 SCE/cell and 49 +/- 8.5% of cells with aberrations respectively. Chromosomal aberrations included multiple complex breakage and rearrangements. In HBSS containing Ca2+ (0.575 mM) and Mg2+ (0.4 mM) in concentrations equivalent to those in RPMI 1640, maximum SCE and aberration responses of 31.8 and 28% were observed in cells treated at pH 6.0. Similarly, maximum SCE frequencies (46 +/- 1.6 SCE/cell) and percentage of cells with aberrations (8 +/- 1.4%) were present in spleen cells treated at pH 6.0 in RPMI media. SCEs and aberrations decreased with increasing pH in either media containing divalent cations. In Ca2+/Mg2(+)-free HBSS, the highest mitotic index and fastest cell cycling were seen at pH 6.0. Mitotic indices dropped dramatically at pH 7.4 but recovered considerably at pH 8.0, in spite of a high frequency of cells containing aberrant chromosomes. The most dramatic cytotoxicity occurred at pH 6.0 in HBSS containing Ca2+ and Mg2+. Decreased cytotoxicity was apparent at higher pH and in RPMI medium. Conditions for optimal growth of control cells were obtained following pulse-treatment in Ca2+/Mg2(+)-free HBSS medium at high pH (8.0). Because of the dramatic cytogenetic toxicity of exogenously supplied epsilon-ATP, and the ubiquitous occurrence and biological importance of intracellular ATP, the latter should be considered a potential target for adduct formation by electrophilic metabolites of carcinogenic agents.

Sister chromatid exchange induced by etheno-ATP derivatives in vitro.

Genotoxic activities of a series of commercially purchased 1,N6-ethenoadenosine (epsilon-Ado) and epsilon-deoxyadenosine (epsilon-dAdo) derivatives were assessed using the sister chromatid exchange (SCE) assay in murine spleen lymphocytes in vitro. Of the epsilon-Ado adducts evaluated for SCE induction epsilon-ATP and epsilon-dATP were highly active (5x baseline) SCE inducers over a concentration range of 50-150 microM. Moderate SCE-inducing activities were seen with epsilon-dAdo, epsilon-A, and epsilon-AMP. epsilon-A was of particular interest in that spleen lymphocytes from a single mouse were highly sensitive to SCE (greater than 50 SCE/cell at 75 microM). epsilon-Ado was weakly effective and epsilon-ADP and epsilon-dAMP did not produce significantly elevated SCEs. Cocanavalin A-stimulated T-lymphocytes and lipopolysaccharide-stimulated B-lymphocytes exhibited comparable SCE responses to epsilon-A, epsilon-AMP, and epsilon-dATP. However, B-lymphocytes were considerably less sensitive than T-lymphocytes to epsilon-dAdo and epsilon-ATP. Evaluation of the purities of specific epsilon-Ado derivatives, as performed by high-performance liquid chromatography and thin layer chromatography, failed to detect potential contaminants as cytogenetically active agents. However, a difference (about threefold) in cytogenetic activities of two lot numbers of epsilon-ATP paralleled the difference in UV absorbance of quivalent concentrations (mg/ml), prepared according to the manufacturers stated purity. Any impurities likely to be present were consistent with inactive nonchromophoric compounds such as buffer salts. Because of the direct genotoxic activity of epsilon-A in intact mammalian cells, we suggest that intracellular adenylate pools, including the prominent ubiquitous nucleotide ATP, are non-DNA targets for epsilon-modification by active metabolites and the resulting epsilon-adducts are likely to be active moieties in SCE induction and in neoplastic transformation produced by ethyl carbamate.

Photoaffinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 8-azido-1,N6-ethenoadenosine 5'-triphosphate.

Steady-state kinetic measurements have shown that 8-azido-1,N6-ethenoadenosine 5'-triphosphate (8-N3-epsilon ATP) can be noncovalently bound to rabbit muscle fructose 1,6-bisphosphate aldolase with Ki = 0.075 mM at pH 8.5. This binding is purely competitive with substrate and occurs at the strong binding site for mononucleotides. Photoaffinity labeling of aldolase in the presence of 8-azido-1,N6-ethenoadenosine 5'-triphosphate results in inactivation of the enzyme. Aldolase is protected against modification in the presence of the inhibitors hexitol 1,6-bisphosphate or ATP. The labeling is saturable, and a good correlation is observed between the loss of enzymatic activity and the incorporation of 8-N3-epsilon ATP into aldolase. In addition, aldolase loses its ability to bind to phosphocellulose following modification. Digestion of labeled protein with trypsin, chymotrypsin, and cyanogen bromide revealed substantial modification of peptide 259-269. Thr-265 was identified as the residue that was covalently modified by 8-N3-epsilon ATP. On the basis of these results and other data we propose a model for the mononucleotide binding site.

Angle of active site of myosin heads in contracting muscle during sudden length changes.

The change in orientation of myosin crossbridges in contracting muscle during sudden length changes was examined by fluorescence polarization. This study used a fluorescent ATP analogue, 1,N6-etheno-2-aza-ATP(epsilon-2-aza-ATP) as a probe. Its fluorescence is considerably enhanced upon binding with myosin and is dependent on the chemical state of the myosin-nucleotide complex in muscle. The results showed that nucleotides bound to crossbridges in the intermediate attached state (presumably AM-epsilon-2-aza-ADP-Pi) during isometric contraction are highly oriented at the same angle as that of AM in rigor with bound epsilon-2-aza-ADP. Furthermore the orientation of nucleotides bound to crossbridges in the attached state is not altered during sudden changes in length of isometrically contracting muscle. The results of this time-resolved measurement support the conclusion obtained from a previous steady-state experiment that change in axial orientation of the active site of the myosin head is not involved in force generation.

Detection of the conformational change in the catalytic site of adenosine triphosphatase from beef liver mitochondria by affinity labeling with the dialdehyde derivative of ethenoadenosine triphosphate.

Beef liver mitochondrial F1ATPase was inactivated by the 2',3'-dialdehyde derivative of ethenoATP (epsilon ATP) in a pseudo-first order reaction. The kinetics of protection of the enzyme against inactivation by various nucleoside triphosphates (NTPs) revealed that the dial-epsilon ATP was bound to the catalytic site as an affinity label. Certain anions (sulfate or bicarbonate) were ineffective for protection. In the early phase of the reaction, inactivation was due to the binding of 1 mol dial-epsilon ATP per mol enzyme. In this phase, dial-epsilon ATP bound exclusively to the subunit beta of the enzyme, indicating that the catalytic site is in this subunit. The fluorescence of the ethenoadenosine moiety, bound exclusively to the subunit beta of the enzyme, was measured as a conformational probe of the catalytic site region. Addition of ATP or CTP to the labeled enzyme resulted in a decrease in the fluorescence intensity. GTP and other NTPs were less effective than ATP or CTP. The anions (sulfate of bicarbonate) suppressed the ability of ATP to decrease the fluorescence in a competitive manner. Quantitative analysis of these fluorescence changes suggested that they might originate from the binding of the NTP to the regulatory site of the enzyme. These findings are in good agreement with the two-site model proposed by us (Wakagi, T. & Ohta, T. (1981) J. Biochem. 89, 1205) which was deduced from the steady state kinetics of the NTPase reactions catalyzed by the F1ATPase.

Amphoteric charge distribution at the enzymatic site of 1,N6-ethenoadenosine triphosphate-binding heavy meromyosin determined by dynamic fluorescence quenching.

The features of the charge distribution in the vicinity of the ATP-binding site of heavy meromyosin (HMM) were investigated by the technique of dynamic fluorescence quenching. Instead of ATP, 1,N6-ethenoadenosine triphosphate (epsilon-ATP), a fluorescent derivative of ATP, was attached to the ATP-binding site in the presence of an ATP-regenerating system. The I- ion and acrylamide were used as negative- and zero-charged quenchers. In addition to these quenchers, we used the TI+ ion, which has recently been found to be a highly efficient quencher with positive charge, and to be generally applicable to fluorescence-labeled proteins. The Stern-Volmer quenching constants of TI+ and I- for epsilon-ATP bound to HMM both decreased with increasing ionic strength of the solvent. This result means that there is an electrostatic attractive force between the fluorophore ad both TI+ and I-. On the other hand, the Stern-Volmer quenching constant of acrylamide was not significantly affected by a change in ionic strength. This result confirms that no significant change in protein conformation in the vicinity of the epsilon-ATP-binding site of HMM occurs with change in ionic strength. In order to interpret these results, we propose a model in which a positive charge is located on one side of the epsilon-adenine ring and a negative charge is located on the opposite side. The negative charge is attributed to the phosphate group in epsilon-ATP and positive one is probably attributable to a lysyl residue in the polypeptide chain of HMM.

The interaction of nucleotides with F1-ATPase inactivated with 4-chloro-7-nitrobenzofurazan.

In common with the F1-ATPase from other sources, yeast mitochondrial F1-ATPase was inhibited by 4-chloro-7-nitrobenzofurazan. Total inhibition of the F1-ATPase activity was compatible with the modification of a single tyrosine residue per F1-ATPase molecule. Radioactive labelling experiments localized this modification on a beta-subunit. The inactive modified enzyme retained the capacity to bind the photoaffinity label 8-azido-1,N6-etheno-ATP, which has previously been shown to bind nucleotide sites of low affinity. As well, the inactive modified enzyme bound MgATP with high affinity, yielding a Kd of 14 microM. The results are consistent with the hypothesis of alternating, or cooperative, site catalysis by F1-ATPase.

Inhibition of mammalian polyadenylate polymerase by 2-aza-1,N6-etheno-adenosine triphosphate.

2-Aza-1,N6-etheno-adenosine triphosphate (aza-epsilonATP), a fluorescent analog of adenosine triphosphate, significantly inhibits polyadenylate [poly(A)] polymerase of bovine lymphosarcoma and calf thymus, with 50% inhibition at 200 muM (in the presence of an equal concentration of adenosine triphosphate). Calf thymus RNA polymerases II and III are inhibited 32 and 20%, respectively, by a 3.8-fold excess of aza-epsilonATP; DNA polymerase alpha is not inhibited. The inhibition of poly(A) polymerase by aza-epsilonATP appears to be competitive with adenosine triphosphate; incorporation of aza-epsilonATP is not observed. Polymers of 2-aza 1,N6-etheno-adenosine monophosphate are used as primers, but pootly. 1,N-Etheno-adenosine triphosphate and 9-beta-D-arabinofuranosyladenine triphosphate are poor inhibitors of poly(A) polymerase; adenosine diphosphate is ineffective. Deoxyadenosine triphosphate inhibits to the same extent as aza-epsilonATP, while other naturally occurring nucleotides inhibit poly(A) polymerase to varying degrees, with deoxynucleoside triphosphates more potent than ribonucleoside triphosphates. Inhibition of poly(A) polymerase by naturally occurring nucleoside triphosphates suggests that nucleotides may regulate the enzyme in vivo; inhibition by the fluorescent analog aza-epsilonATP suggests that this compound may be useful in elucidating poly(A) metabolism in both normal and neoplastic cells.