Electron-microscopic studies on location of SH-groups in mitochondrial F1-ATPase using a ferritin label.

A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in alpha, gamma or epsilon subunits), it is inferred from the results obtained that one of the protein masses is a complex between beta subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F1-F0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F0 component is bound to two adjacent protein masses of the F1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.

Two distinct types of SH-groups are necessary for bumetanide and bile acid uptake into isolated rat hepatocytes.

Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 microM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [3H]bumetanide and [3H]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic abd interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 microM, DTONB 141 microM) and for cholate uptake (DTSNB 250 microM, DTONB 115 microM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.

Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent.

From 1,9-nonylenedithiol and Ellman's Reagent the bifunctional asymmetric disulfide n-nonylene-1,9-bis-[5-dithio-(2-nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine-374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2-nitro-5-thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys-374-Cys-374') as well as a monomer with a crosslink between Cys-374 and Cys-10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol-specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.

Demonstration of the feasibility of observing nuclear magnetic resonance signals of 77Se covalently attached to proteins.

Previous 77Se NMR relaxation time studies established the utility of 77Se NMR spectroscopy in studying low molecular weight (less than 500) selenium-containing molecules. Since the spin rotation and chemical shift anisotrophy mechanisms contributed significantly to the 77Se spin-lattice relaxation in these compounds, it was questionable as to whether the latter mechanism would be efficient enough to enable 77Se resonances to be observed in a reasonable period in high molecular weight selenobiomolecules. Thus, to address this problem, disulfide bonds of ribonuclease-A and lysozyme were reductively cleaved under denaturing conditions, and the resulting 7-8 sulfhydryl groups were treated with a new sulfhydryl group reagent containing selenium, 6,6'-diselenobis(3-nitrobenzoic acid), to give proteins containing covalently attached selenium in the form of selenenyl sulfides. The observation of high resolution 77Se NMR spectra of these proteins under denaturing conditions was accomplished. Five to six 77Se NMR resonances, which fell in a chemical shift range of 14-15 ppm, were observed for each protein and are compared to the chemical shifts of several model selenenyl sulfides derived from cysteine.

Solubilization of hydrophobic peptides by reversible cysteine PEGylation.

"PEG-a-Cys" reagent, synthesized by the esterification of monomethoxy-poly(ethylene glycol) (avg. MW = 5 kDa) to Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], is shown to "PEGylate" reversibly the cysteine residue of a 25-residue synthetic hydrophobic peptide (H2N-REAAALAAAAALAAWAALCPARRRR-CO2H) designed to model a transmembrane segment of a membrane protein. A mixed disulfide bond was formed between the reagent and the peptide that was readily cleaved with the mild reducing agent tricarboxyethylphosphine hydrochloride (TCEP.HCl). Carboxypeptidase B digestion of the charged carboxyl terminus of the peptide through to the Ala residue--which mimics the enzymatic cleavage of a TM segment from a fusion protein--releases a highly hydrophobic peptide. A time-dependent decrease in the amplitude of the digested peptide circular dichroism (CD) spectra was attributed to the aggregation and/or precipitation of the peptide. While PEGylation of the peptide with PEG-a-Cys had a negligible effect on conformation, it inhibited the loss of CD amplitude in both intact and digested peptides, suggesting that it was effective in solubilization of hydrophobic peptides.