A. Di Pietro

IF1 inhibition of mitochondrial F1-ATPase is correlated to entrapment of four adenine- or guanine-nucleotides including at least one triphosphate

This paper demonstrates that the inhibition of F1 ATPase activity by the natural inhibitor protein IF1 is correlated to triphosphate nucleotide entrapment in F1. The complete balance of nucleotides bound after preincubation with Mg-[alpha-32P]GTP or Mg-[alpha-32P]ATP, used to promote IF1 inhibition, has been established on purified F1 containing 0.7 mol of non-exchangeable endogenous nucleotides. As many as 4 mol of labelled guanine- or adenine- nucleotides are trapped in F1; at least one of these nucleotides is a triphosphate. On the contrary, in the absence of IF1, no triphosphate nucleotide is significantly retained and the diphosphate nucleotides bound are mainly exchangeable.

Prenylated Xanthones as Potential P-Glycoprotein Modulators

Dimethylallyl (DMA) derivatives of a naturally occurring xanthone (decussatin 1) were prepared. Their activity as potential P-glycoprotein inhibitors was monitored by affinity of direct binding and compared to that of corresponding DMA-flavones. Both classes of compounds exhibited the same structure-activity relationships. Decreasing polarity enhanced the binding affinity for the P-glycoprotein C-terminal cytosolic domain since DMA derivatives were more active, but unsubstituted hydroxyl group close to the carbonyl was required for efficient activity.

The ATP-binding Site in the 2-Kinase Domain of Liver 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase STUDY OF THE ROLE OF Lys-54 AND Thr-55 BY SITE-DIRECTED MUTAGENESIS

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding. We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine. Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type. The mutations had no effect on fructose-2,6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3′,5′-AMP-dependent protein kinase. Binding experiments with ATP, ADP, and their analogs (3′-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role. Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.

Competition between ADP and nucleotide analogues to occupy regulatory site(s) related to hysteretic inhibition of mitochondrial F1-ATPase

Abstract The regulatory site(s) responsible for ADP-induced hysteretic inhibition of pig heart mitochondrial F 1 -ATPase appeared to be specific of adenine nucleotides. The site(s) cannot be readily occupied by guanosine analogues although GTP is hydrolyzed at the catalytic sites. The length of the phosphate chain must be that of a nucleoside-diphosphate. Adenosine β,γ-imidotriphosphate, dialdehyde derivative of ADP also bind to the site(s) while ribosering opened analogues do not. It is also demonstrated that saturation of only one site, specifically by ADP, might be sufficient to induce hysteretic inhibition. However it cannot be excluded that other site(s), less specific, must also be saturated by nucleotides to permit ADP-inhibitory effects.

Masking of co-operativity of nucleotide sites in pig heart mitochondrial ATPase (F1) by heating

Although the soluble mitochondrial ATPase (F,) [l] is an enzyme complex constituted of 5 polypeptides with different stoichiometry, most studies report linear Michaelis and Menten kinetics [2-41 for ATP hydrolysis and its inhibition by ADP. Kinetic studies did not reveal several nucleotide sites in purified F,-ATPases** except in the case of rat liver ATPase by Ebel and Lardy [S] who observed a concave curvature of reciprocal plots of initial velocity with MgATP as the variable substrate, in the absence of other anions. In a previous paper [6], we applied the Senior and Brooks procedure [7] to purify pig heart mitochondrial ATPase (Fi). The resulting preparation behaved mainly as that of beef heart in other laboratories (cf reviews from Senior [8], Pedersen [9] and Penefsky [lOI 1. The present work shows that with soluble pig heart mitochondrial ATPase (F,), prepared without the heating step, interaction between ATP and ADP sites can be demonstrated by kinetic studies: reciprocal plots of initial velocity with ATP as the variable substrate, become curved with increasing ADP concentrations (Hill coefficient from 1 to 2.1). This

A yeast strain with mutated beta-subunits of mitochondrial ATPase-ATPsynthase: high azide and bicarbonate sensitivity of the ATPase activity

A phenotypic revertant with modified beta-subunits of mitochondrial ATPase-ATP synthase has been obtained for the first time by selection from a beta-less mutant of the yeast Schizosaccharomyces pombe. Contrary to the parental mutant, the phenotypic revertant grows on glycerol, has normal respiratory activity and shows immunodetectable beta-subunits. However the kinetic properties of its submitochondrial particles ATPase activity differ markedly from those of the wild strain. The optimal pH is increased by about one unit. The maximal rate of the revertant ATPase activity at pH 8.5 is 4 to 5-fold lower than that of the wild strain, but it can be greatly increased upon addition of bicarbonate whereas the wild strain is completely insensitive to this anion. Furthermore the revertant ATPase activity is much more sensitive to azide inhibition. The results suggest that ADP dissociation is the rate-limiting step of ATP hydrolysis by the revertant.

Revertant of the yeast Schizosaccharomyces pombe with modified alpha subunits of mitochondrial ATPase-ATPsynthase: impaired nucleotide interactions with soluble and membrane-bound enzyme

A partial revertant from a mutant with modified alpha subunits of mitochondrial ATPase-ATPsynthase has been obtained for the first time from the yeast Schizosaccharomyces pombe. The purified F1 contains a lower amount of endogenous nucleotides as compared to the wild-strain enzyme. In contrast to the wild-type, the F1 ATPase activity from the revertant does not exhibit bicarbonate-sensitive negative cooperativity. The revertant Michaelis constant for Mg-ATP is very similar to that of normal F1 in the presence of bicarbonate while the Vm is slightly lower. The revertant enzyme is much less sensitive to inhibitions by ADP and by azide. It is proposed that the lack of negative cooperativity of revertant F1 ATPase activity is due to lower affinity for ADP, the release of which is no longer the rate-limiting step.

Vicinal dithiol in pig heart mitochondrial F1-ATPase related to thermal or ATP-dependent conformational changes.

Active F1-ATPase prepared from pig heart mitochondria can react with about 2 mol of DTNB (5,5′-dithiobis-2-nitrobenzoic acid) or CPDS (6,6′-dithiodinicotinic acid). The reactivity of these thiol reagents decreases if ATP is absent or if F1-ATPase has been submitted to thermal treatment that increases the specific activity without eliminating any contaminating protein. Affinity chromatography on a Sepharose-DTNB column has shown that the thermal treatment of F1-ATPase induces a conformational change of the enzyme that completely prevents it from being retained on the column while the normal active enzyme can be specifically bound to the Sepharose-DTNB column.A comparative study of the thiols of F1-ATPase reacting with CPDS measured by spectrophotometric estimation of the thione released from CPDS and by [14C]CPDS binding to F1-ATPase suggests involvement of a vicinal dithiol in active F1-ATPase. After CPDS reaction, this vicinal dithiol may become an internal disulfide bridge.

Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.