Effects of ATP analogs on the proton pumping by the vacuolar H+-ATPase from maize roots

Abstract

I. 1994. Effects of ATP analogs on the proton pumping by the vacuolar H*-ATPase from maize roots Understanding the regulatory properties of the activities of the 'V-type adenosine triphosphatase (ATPase) on tonoplast memhranes is important in determining the mechanisms by which this enzyme controls cytoplasmic and vacuolar pH. The possible existence of a regulatory site for adeniae nucieotides was examined by comparing the effects of ADP, adenylylimidodiphosphate (AMP-PNP) and 3'-o-(4-benzoy]) benzoyladenine 5'-triphosphate (BzATP) to those of the 2',3'-dialdehyde derivative of AMP (oAMP) and ATP by using highly purified tonoplast vesicles from maize (Zea mays L. cv, FRB 73) roots. The addition of either .^MP-PNP or BzATP reversibly inhibited the initial rate of proton transport catalyzed by the H*-ATPase in a concentration-dependent manner. Le.ss than 20 y.M AMP-PNP or 50 pM BzATP was, sufficient to inhibit half the initial rate of proton transport in the presence of 2 mM ATP and an excess of Mg, Both analogs increased the K,,, for ATP and reduced the maximum enzyme velocity. The presence of ADP also inhibited proton transport. The characteristics of ADP-induced inhibition were similar to those of BzATP and AMP-PNP. The addition of the periodated derivative of AMP (oAMP) irreversibly inhibited the ATPase in a concentration and time-dependent manner similar to that reported previously (Chow et al. 1992, Plant Physiology 98: 44-52). Irreversible inhibition hy oAMP reduced the maximum velocity of the tonoplast ATPase and was prevented by the addition of ATP. The presence of ADP, AMP-PNP or BzATP had no effect on irreversible inhibition by oAMP The effects of ADP, AMP-PNP and BzATP on the kinetics of ATP utilization and the lack of protection against inhibition by oAMP argue in favor of at least two types of nucleotide binding sites on the V-type ATPase from maize root tonoplast membranes.

The Effect of Trinitrophenyl Nucleotide Derivatives on the Adenosine Triphosphatase Activities in Maize Tonoplast Vesicles

“…Vmax was decreased by approximately 60% and 75 % , respectively obtained in the absence of TNP-ATP. Therefore, the inhibition of proton transport of vacuolar H + -ATPase by TNP-ATP was similar to that observed with ADP inhibition as reported by Brauer and Tu (1994).…”

“…1987,1990,1995) , Hsu cr ul. (1993, 1094) and Brauer and Tu (1994) in nature. The differences in Sensitivity and inhibition of the transport activity and ATP hydrolysis observed in maize root tonoplast vesicles support the indirect mechanism.…”

“…It is of interest to examine the potency of TNP derivatives (i.e., TNP-ATP, TNP-ADP and TNP-AMP) in the inhibition of the ATP hydrolysis. Tu et al (1987Tu et al ( , 1990Tu et al ( , 1995 and Brauer and Tu (1994) showed that the sensitivity and characteristic inhibition of the transport activity and ATP hydrolysis from maize root tonoplast vesicles can be different. The presence of 0.25 mM of TNP-ATP in the normal assay containing 2 mM ATP inhibited 70% of the proton transport activity, while ATP hydrolysis was only reduced by 40%.…”

“…In plants, allosteric modulation (E,E,-type) of ion translocating ATPase by multiple binding of substrate has also been observed. For example, the regulatory effects of ADP on corn root plasma membranes has demonstrated that it may contain more than one binding site for ADP (Brauer and Tu 1994) and the presence of ADP may cause the root plasma membrane ATPase to be less sensitive to the wide range of inhibitors (Tu e? al.…”

“…, 1990Brauer e? d. 1988) on tonoplast membranes, regulation of the activities of V-type ATPase by adenosine nucleotides is favored. Previous studies by Brauer and Tu (1994) demonstrated that several ATP analogs such as ADP (adenosine diphosphate), AMP-PNP (adenylylimidodiphosphate) or BzATP (3'-04benzoyl-benzoyladenosine-5 ' triphosphate) inhibited the kinetics of ATP utilization on the proton pumping of vacuolar H + -ATPase from maize roots. Their results favored at least two types of nucleotide binding sites on the V-type ATPase from maize root tonoplast membranes.…”