Interaction of Cibacron Blue F3GA with glutamine synthetase: use of the dye as a conformational probe. 1. Studies using unfractionated dye samples

Cibacron Blue F3GA dye has been used to probe subtle conformational changes in protein structure associated with the conversion of Escherichia coli glutamine synthetase (GS) between relaxed, taut, oxidized, and dissociated forms. Binding of the dye to each form of the enzyme elicits a different spectral perturbation of the dye which can be detected by difference spectroscopy. By following time-dependent changes in the difference spectrum associated with the binding of dye to the enzyme, it was demonstrated that dissociation of subunits provoked either by urea or by relaxation of the enzyme at pH 8.5 is a multiphasic process. In the presence of 3-4 M urea, dissociation of taut GS is associated with an almost instantaneous, transient increase in absorbancy of the difference spectrum at 638 nm and, after a lag, by a progressive decrease in absorbancy at 585 nm and an increase at 700 nm. The kinetics of these changes vary as a function of temperature, pH, and the concentrations of KCl, MnCl2, and urea, probably reflecting differences in the rates of GS relaxation and in the formation of aggregates of intermediate sizes. Results of direct binding measurements show that the taut and relaxed forms of GS can bind only 1-1.3 equiv of dye per subunit, whereas dissociated subunits bind up to 3.0 equiv per subunit. The Kd of the dye-taut GS complex as calculated from binding data was 0.55 microM. The binding of dye to taut GS was inhibited by its substrate, ADP, and by the allosteric effectors AMP and tryptophan. On the basis of the abilities of ADP, AMP, and tryptophan to inhibit the binding of dye to GS, dissociation constants of the respective GS-ligand complexes were 2.4, 121, and 1170 microM, respectively, in good agreement with previously determined values. From the difference spectra obtained between a given concentration of dye in a 5.0-cm cell and 10 times that concentration in a 0.5-cm cell, it was established that at concentrations greater than 5 microM a significant fraction of the dye is present as stacked aggregates. Because only the dye monomer binds to GS, the difference spectrum between dye and dye bound to GS is due in part to GS-promoted shifts in the equilibrium between stacked and unstacked dye molecules. Consequently, with increasing dye concentrations, the amplitude of the dye vs. dye + GS difference spectrum can continue to increase, even after the GS becomes saturated with dye.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation-specific monoclonal antibodies to glutamine synthetase in Escherichia coli

Glutamine synthetase from Escherichia coli is composed of 12 identical subunits and exists in various forms: unadenylylated, adenylylated, divalent cation bound (taut), and divalent cation free (relaxed). The relaxed dodecamer readily dissociates into monomers upon exposure to 1 M urea or pH 8.0. Glutamine synthetase can be inactivated irreversibly by oxidizing a particular histidine residue or by incubating with methionine sulfoximine and ATP. In order to establish hybridoma monoclones that produce antibodies capable of differentiating between different conformers of glutamine synthetase, homogeneous antibodies produced from 7 clones (10-76-1, 48-76-1, 68-2-1, 57-142-2, 72-104-1, 68-3-2, 57-8-1) were characterized for their binding specificity and effects on glutamine synthetase activity. Two antibodies (10-76-1, 48-76-1) bind only to the monomeric form, two antibodies (57-142-2, 68-3-2) bind only to the dodecameric forms (taut or relaxed) and the three others (68-2-1, 72-104-1, 57-8-1) bind to both forms. At a low antibody concentration, 68-3-2 binds preferentially to taut glutamine synthetase over oxidized glutamine synthetase. None of the 7 antibodies differentiates between unadenylylated and adenylylated form. Nevertheless, the gamma-glutamyltransferase activities of the resulting antibody-glutamine synthetase complexes were influenced variably depending upon the state of adenylylation and the divalent cation.     

Isolation and partial characterization of the major apolipoprotein component of bovine serum high density lipoprotein.

The delipidated protein component of bovine serum high density lipoprotein was fractionated by gel filtration on a Sephadex G-150 column (equilibrated with buffer containing 6 M urea) into three fractions: I, II and III. Fractions I and II together constitute 88% of all the protein weight of bovine high density lipoprotein, whereas fraction III accounts for the remaining 12%. Analysis of the fractions by sodium dodecyl sulfate-polyacrylamide electrophoresis reveals that fraction I consists mostly of aggregated forms of fraction II and some higher molecular weight species, probably irreversible aggregates of fraction II. The irreversible aggregates are apparently formed during the delipidation procedure or upon aging of the lipoprotein. The major protein component of the high density bovine lipoprotein is found in fraction II; it has a molecular weight of 27 000 plus or minus 1500 and appears to be homogeneous by several physicochemical criteria. The amino acid composition of fractions I and II are essentially identical; their spectral properties, including absorption, fluorescence, and circular dichroism spectra, are similar; however, fraction I appears to contain traces of oxidized lipid and more secondary alpha-helical organization than fraction II. By comparison with the intact lipoprotein, which contains about 65% of alpha-helical structure, fractions I and II have diminished alpha-helical organization, 55% and 43%, respectively. Fraction III, on sodium dodecyl sulfate-polyacrylamide electrophoresis, separates into two protein bands of equivalent intensity, having molecular weights around 13 000 and 11 000. Fraction III is markedly distinct from the other two, in amino acid composition and spectral properties, especially in its red-shifted fluorescence and very low content of alpha-helical structure. The protein composition of bovine serum high density lipoprotein is compared with recently published results for high density lipoprotein apoproteins of man, chimpanzee, rhesus monkey, pig and rat. Similarities and differences are discussed in terms of possible evolutionary and functional factors.     

Separation and characterization of receptor-translocation inhibitors from AH 130 tumor cells

The objective of this investigation was to study the relationship between glucocorticoid resistance and macromolecular receptor-translocation inhibitors ( MTIs ). MTIs in various cytoplasmic preparations are known to inhibit the "activated" receptor-steroid complex association with isolated nuclei, chromatin, or DNA. It was found that the MTI in the cytosol of AH 130 tumor cells (glucocorticoid resistant cells) appeared to be about 5 times more inhibitory than crude MTI from rat liver. Another difference between these MTI preparations was that ATP decreased the inhibition by crude MTI from rat liver, but had little effect on that of MTI from the tumor cells. Both preparations gave three fractions of material with inhibitory activity on DEAE-cellulose chromatography. The first fraction (Peak I), eluted with about 0.1 M NaCl, was the largest fraction separated from the tumor cytosol, but a minor fraction of that from liver. In the presence of 5 mM ATP, Peak I from rat liver enhanced nuclear binding, but that from the tumor did not, suggesting that these fractions were qualitatively different. The other two fractions (Peak II and Peak III), eluted with about 0.2 M and 0.3 M NaCl, respectively, were comparable in the two preparations.     

Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.     

Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.     

Some chemical, physical and histochemical properties of three diamine fractions obtained by gel chromatography from the high-iron diamine staining solution used for localizing sulphated mucosaccharides

Synopsis The coloured components in the high-iron diamine dye bath were separated into three fractions using column chromatography on Sephadex G-10. These fractions were called Fraction I, II and III in order of their emergence from the column. From atomic absorption measurements, part of Fraction I was found to be free of iron. Most of Fraction II and the whole of Fraction III contained only trace amounts of iron. Therefore, the three Fractions were investigated further. All experiments were carried out at pH 1.4 (corresponding to the pH of the original high-iron diamine bath).Fraction I was violet, Fraction II red-violet and Fraction III aniline-red; the extinction maxima in the visible region were 560, 526 and 540 nm respectively. On electrophoresis, the Fractions were not quite homogeneous, although most of Fractions I and II migrated in the same front and much faster than Fraction III. The high-iron diamine solution separated into two main fractions, one of which corresponded in colour and velocity to Fractions I and II and the other to Fraction III.In histochemical experiments, Fractions I, II and III bound to tissue sites containing sulphated mucosaccharides or nucleic acids; from histochemical enzyme digestion tests or by using purified materials in spot tests on cellulose acetate membrane, it was confirmed that the diamines were bound to RNA and DNA. However, when ferric chloride was added to any of the Fractions in an amount corresponding to that in the original high-iron diamine dye bath, the binding to tissue sites containing nucleic acids was inhibited but the reaction with sulphated mucosubstances was not affected. Also, in the presence of added ferric chloride, the anomalous binding of Fraction I to carboxyl groups of mouse sublingual gland sialomucin was prevented.It is concluded that ferric chloride in the high-iron diamine dye bath prevents diamine complexes from binding with nucleic acids, and apparently with carboxymucins too. Further, this conclusion substantiates our previous observations of the central role ferric chloride plays in making the histochemical high-iron diamine technique specific for sulphated mucosubstances.     

Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile.

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.     

Mode of action of cellulases on dyed cotton with a reactive dye

Cotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases.     

Competitive cooperative bindings of a small ligand to a linear polymer. II. Investigations on the mechanisms of proflavine binding to poly (A) and DNA.

Experimental binding isotherms relative to the interactions between proflavine and poly(A) or DNA are analyzed by comparison with theoretical models dealing with competitive cooperative bindings. In the case of poly(A), there are apparently no specific binding sites for the positive co-operative binding (complex I) leading to dye aggregation along the polyanionic chain. The second complex (complex II) seems to involve specific base-dye interactions, but it cannot be said whether this binding displays negative cooperativity or noncooperativity. None of the two simpler theoretical models agree quantitatively with all experimental data. A plausible interpretation can be given if it is assumed that (i) the electrostatic binding of one isolated bound dye molecule (nucleus of complex I) involves a definite interaction between a phosphate group and the positive charge of the dye; (ii) the structure of complex II is such that a dye–phosphate ionic interaction is maintained. In the case of DNA, our model of monoexclusive interactions fits the data more closely than does the model of biexclusive interactions. This gives experimental support for structural models in which the intercalated molecule interacts preferentially with one strand of the double helix and blocks only one phosphate for electrostatic binding. In order to propose a mechanism consistent with equilibrium and relaxation kinetic data, a modified reaction scheme is considered which takes account of the cooperativity effects in external binding and extends previous models.     

Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle

The binding of calcium by isolated sarcoplasmic reticulum from cow uterus was studied. Sarcoplasmic reticulum was prepared by differential centrifugation. Three fractions were obtained: I, sedimented between 2,500–15,000 x g; II at 40,000 x g; and III, at 150,000 x g. Fraction II was further purified on a sucrose density gradient. All three fractions contained considerable amounts of intrinsic calcium, mostly in fraction I. Calcium binding in the presence of ATP¹ and Mg also was greatest in fraction I, followed by fraction II, with less in fraction III. Without ATP no calcium was taken up. 5 and 10 mM sodium azide partially inhibited calcium binding in fraction I, but not in fraction II, suggesting the presence of some mitochondria or mitochondrial fragments in fraction I. Calcium binding in fraction II was completely inhibited by 3 mM salyrgan; this fraction thus appears to be sarcoplasmic reticulum. ATPase activity was found in all three fractions, highest in fraction II. It is computed that calcium binding in fractions I and II, on the basis of a 50% yield of protein, is sufficient to elicit contraction by supplying calcium to the contractile proteins of the smooth muscle cell and to regulate relaxation and contraction.     

Hepatic microsomes from freshwater fish--I. In vitro cytochrome P-450 chemical interactions

1. Of 87 chemicals tested for their ability to interact with oxidized hepatic cytochrome P-450 from mature male brook trout (Salvelinus fontinalis), 21 formed detectable type I or type II binding spectra. 2. When 8 of these 21 chemicals were tested with cytochrome P-450 of nine other species of freshwater fish, wide species variation in hepatic microsomal cytochrome P-450 was evident, since the spectral size of chemical interactions as related to the carbon monoxide spectrum and the ratio of type II to type I binding were not alike. 3. These spectral data suggest that hepatic microsomal cytochrome P-450 of freshwater fish exists in different forms.     

Thermodynamics of mucopolysaccharide–dye binding. II. Binding constant and cooperativity parameters of acridine orange–dermatan sulfate system

In the acridine orange–dermatan sulfate system, free and bound dye can be distinguished from each other spectroscopically. This permits the use of fluorometric methods to study the binding of acridine orange to the acid mucopolysaccharide dermatan sulfate. Experiments were conducted at 24°C in 10^?3 M citrate/phosphate buffer at pH = 7.0. The binding of the dye is highly cooperative, as evidenced by considerable interaction between adjacent bound dye molecules. Analysis of the data indicates that dermatan sulfate binds 2.3 ± 0.3 mol of acridine orange per dermatan sulfate uronic acid residue with a cooperative binding constant, K_q ranging from 4.9 to 6.0 × 10⁵ M^?1 which corresponds to a free energy of 7.74 ? ΔG° ? 7.86. The cooperativity parameter q apparently increases with increasing polymer-to-dye ratio.     

Thermodynamic effects of active-site ligands on the reversible, partial unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric studies.

Dodecameric glutamine synthetase (GS) from Escherichia coli undergoes reversible, thermally induced partial unfolding without subunit dissociation. A single endotherm for Mn.GS (+/- active-site ligands) in the presence of 1 mM free Mn2+ and 100 mM KCl at pH 7 is observed by differential scanning calorimetry (DSC). Previous deconvolutions of DSC data for Mn.GS showed only two two-state transitions (with similar tm values; 51.6 +/- 2 degrees C), and indicated that cooperative interactions link partial unfolding reactions of all subunits within the Mn.enzyme dodecamer [Ginsburg, A., & Zolkiewski, M. (1991) Biochemistry 30, 9421]. A net uptake of 8.0 equiv of H+ by Mn.GS occurs during partial unfolding, as determined in the present DSC experiments conducted with four buffers having different heats of protonation at 50 degrees C. These data gave a value of 176 +/- 12 kcal (mol of dodecamer)-1 for delta Hcal corrected for buffer protonation. L-Glutamine and L-Met-(SR)-sulfoximine stabilize the Mn.GS dodecamer through the free energies of ligand binding, and these were shown to be partially and totally released, respectively, from the 12 active sites at high temperature. Ligand effects on Tm values from DSC were similar to those from spectral measurements of Trp and Tyr exposures in two subunit domains. Effects of varying [ADP] on DSC profiles of Mn.GS were complex; Tm is increased by low [ADP] and decreased by > 100 microM free ADP. This is due to the exposure of an additional low-affinity ADP binding site per GS subunit at high temperature with log K1' = 4.3 and log K2' = 3.6 at 60 degrees C relative to log K' = 5.5 for ADP binding at 30 degrees C, as determined by isothermal calorimetric and fluorescence titrations. Moreover, delta Hcal at > 27% saturation with ADP (corrected for ADP binding/dissociation) is approximately 80-100 kcal/mol more than in the absence of ligands. Changes in domain interactions could result from ADP bridging subunit contacts in the dodecamer. Each of the active-site ligands investigated here produces different effects on DSC profiles without uncoupling the extremely cooperative, partial unfolding reactions in the Mn.GS dodecamer.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.     

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography.

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Surface potential on purple membranes and its sidedness studied by a resonance Raman dye probe.

A new technique for the measurement of membrane surface potential is proposed and demonstrated. The method is based on the fact that a positively charged styryl dye molecule aggregates when present at high concentration in the Debye layer near a membrane bearing a negative surface potential. The dye in its aggregated form exhibits marked differences in its resonance Raman spectrum relative to the free dye molecules. This method was used to study the potential on the surfaces of the purple membrane that contains the pigment bacteriorhodopsin. A value of -29.5 mV was found for membranes with bacteriorhodopsin in its relaxed, light-adapted state, and the potential decreased to -34.5 mV when most of the bacteriorhodopsin was converted to the M412 intermediate. Because the dye probe does not diffuse through the lipid bilayer, it can be used to probe the potential on the external or internal surface of a vesicle. Thus, we found that the potential on the purple membrane was asymmetric and was localized mainly on the surface that faces the cytoplasm in the cell.     

EPR and electron nuclear double resonance investigation of oxidized hydrogenase II (uptake) from Clostridium pasteurianum W5. Effects of carbon monoxide binding

Two different hydrogenases have been isolated from Clostridium pasteurianum W5. Hydrogenase II (uptake) is active in H2 oxidation while hydrogenase I (bidirectional) is active both in H2 oxidation and evolution. Previous EPR and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I have now been complemented by analogous studies on oxidized 57Fe-enriched hydrogenase II and its CO derivative (using 12CO and 13CO). Binding of CO greatly changes the EPR spectrum of oxidized hydrogenase II, and use of 13CO leads to resolved hyperfine splitting from interaction with a single 13CO molecule (AC approximately 34 MHz). This coupling is over 50% larger than that seen for hydrogenase I. 57Fe ENDOR disclosed two types of iron site in both oxidized hydrogenase II and its CO derivative. Combination of EPR, ENDOR, and M?ssbauer results shows that site 1 has AFe1 = 18 MHz shifting to approximately 30 MHz upon CO binding and consisting of two Fe atoms and site 2 has A2 approximately 7 MHz shifting to approximately 10 MHz and containing a single Fe. These results are very similar to those seen for hydrogenase I, which indicates that a structurally similar 3Fe cluster, believed to be the catalytically active site, is present in both. Proton ENDOR shows a solvent exchangeable resonance only in the CO derivative of hydrogenase II. This indicates a structural difference between hydrogenases I and II that is brought out by CO binding. No evidence of 14N coordination to the cluster is seen for either enzyme.