Bifunctional Thiol Research Articles

The γ subunit of F 1 and the PVP protein of F o (F oI) are components of the gate of the mitochondrial F oF 1 H +-ATP synthase

The γ subunit of the F 1 moiety of the bovine mitochondrial H +-ATP synthase is shown to function as a component of the gate. Addition of purified γ subunit to F o-liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the γ subunit with the PVP protein of the F o sector.

Sulfhydryl Reagents and Energy-Linked Reactions in Monocot Thylakoids

Monofunctional maleimides have been used to covalently modify the coupling factor protein of monocot thylakoid membranes. As with dicot thylakoids, incubation of the monocot thylakoids with maleimides in the light but not in the dark results in inhibition of both ATP synthesis and hydrolysis. In the dark, sites on the gamma and epsilon subunits of maize Zea mays coupling factor 1 are modified after incubation of maize mesophyll thylakoids with the fluorescent maleimide N-(anilinonaphthyl-4) maleimide. A light accessible site localized solely to the gamma subunit has also been demonstrated. In contrast to the case with dicot thylakoids (spinach [Spinacia oleracea] and pea [Pisum sativum]) treatment of monocot thylakoids (maize, barley [Hordeum vulgare], crabgrass [Digitaria sanguinalis]) with bifunctional maleimides or thiol oxidants in the light does not result in functional uncoupling, i.e the bifunctional reagents act more like energy transfer inhibitors. The lack of functional uncoupling could be due either to a failure of the reagents to cross-link key sulfhydryl residues in the gamma subunit or to the continued ability of the gamma subunit to gate proton movements through the chloroplast coupling factor complex even though its conformation has been altered by sulfhydryl reagents.

UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification.

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1.

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.

Effect of diamide on proton translocation by the mitochondrial H +-ATPase

Treatment of sonic submitochondrial particles with the bifunctional thiol reagent, diamide, results in an enhancement of proton conductivity and ATPase activity, which is reversed by the reducing agent dithiothreitol, is suppressed by F 0 inhibitors like oligomycin and is absent in particles that are deprived of peripheral F 0 polypeptides. The effect of diamide is apparently due to oxidation of dithiols to disulfides in peripheral polypeptide(s) of F 0.

A comparison of catecholamine-induced internalization of beta-adrenergic receptors and receptor-mediated endocytosis of epidermal growth factor in human astrocytoma cells. Inhibition by phenylarsine oxide.

The ligand-induced internalization of beta-adrenergic receptors and the receptor-mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups.

Photochemical probes of the active site of myosin. Irradiation of trapped 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate labels the 50-kilodalton heavy chain tryptic peptide.

3'-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP), an analog of ATP containing a photoreactive benzophenone moiety, was used as a probe of the ATP binding site of myosin subfragment 1 (SF1). The inactivation of SF1 NH+4-EDTA ATPase by the bifunctional thiol crosslinking system cobalt(II)/cobalt(III) phenanthroline complexes was enhanced by Bz2ATP to the same degree as by ATP. This treatment resulted in the stable trapping of Bz2ATP at the active site in nearly stoichiometric amounts in a manner exactly analogous to ATP (Wells, J.A., and Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970). Irradiation of SF1 containing trapped [3H]Bz2ATP gave approximately 50% covalent incorporation of the trapped nucleotide into the enzyme. Analysis of photolabeled SF1 by gel electrophoresis showed that all of the [3H]Bz2ATP was attached to the 95-kDa heavy chain fragment. No label was found in the light chains. Similar analysis of the same protein after limited trypsin treatment demonstrated that approximately 75% of the [3H]Bz2ATP was bound to the central 50-kDa peptide and its 75-kDa precursor from the heavy chain. The N-terminal 25-kDa tryptic peptide, shown to be photolabeled by other ATP analogs (Szilagyi, L., Balint, M., Sreter, F.A., and Gergely, J. (1979) Biochem. Biophys. Res. Commun. 87, 936-945; Okamoto, Y., and Yount, R.G. (1983) Biophys. J. 41, 298a), was not labeled (less than 1%) by Bz2ATP. These results demonstrate that portions of the 50 kDa-peptide of the heavy chain are within 6-7 A of the ATP binding site on SF1 and possibly contribute to nucleotide binding.

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.

Multifunctional cross-linking reagents. I. Synthesis and properties of novel photoactivable, thiol-directed fluorescent reagents.

Bifunctional photo-activable fluorescent thiol reagents of a new type were synthesized. A maleimide group was bonded to an azidocoumarin group via a methylene chain as a spacer. Reagents of this type react first with a cysteine residue of a protein through the maleimide group, and then form another bond with an amino acid side chain of the protein upon irradiation with light, through a nitrene group formed from the azide. Although the reagent is non-fluorescent, the products are highly fluorescent. The fluorescence characteristics of model compounds of these reagents are also described.

On the question of co-operative interaction of myosin heads with F-actin in the presence of ATP

The actin-activated enzymic activity of partially modified heavy meromyosin molecules has been compared to the unmodified protein in order to examine the possibility of co-operative interactions between the two myosin heads. The modification of the myosin heads with the bifunctional thiol reagent p-phenylenedimaleimide proceeded at the same rate with subfragment-1 preparations containing either the alkali 1 chain or alkali 2 chain. The modified, inactivated heads did not bind actin. Enzymic assays of the reacted heavy meromyosin solutions, combined with the analysis of their sedimentation in the presence of F-actin, verified that the two heads reacted with p-phenylenedimaleimide independently. Well-defined heavy meromyosin samples containing fractions of zero, one and two inactivated heads were then examined for their interaction with F-actin in the presence of ATP. Maximum ATP turnover rates, Vmax, were found to be linearly dependent upon the total fraction of unmodified heads and independent of the relative fraction of the one-head modified molecules. The apparent dissociation constants of actin from heavy meromyosin, Kapp, increased somewhat with the increasing fraction of one-head modified molecules. These results are consistent with an independent binding of myosin heads to actin in the presence of ATP.

Comparaison de l'action catalytique de thiols monofonctionnels et de certains thiols bifonctionnels (les mercaptoalcoyl-2 benzimidazoles) sur l'hydrolyse de l'acetate de p-nitrophenyle

Synthesis of bifunctional thiols (2-alkylmercaptobenzimidazoles) and kinetic studies of the catalysis of the hydrolysis of p-nitrophenylacetate by these compounds, and by seven thiols indicates high activity of the 2-mercaptomethylbenzimidazoles in their neutral form, interpreted as the result of nucleophilic catalysis by the thiol function in its neutral state combined with general basic catalysis by the heterocycle.

The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles.

The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether-protein compound with [(14)C]-cysteine shows that 70-80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/10(6)g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/10(6)g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2-4mol of thiol groups/10(6)g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning.