Fluorescence properties of Neurospora tyrosinase.

Some structural properties of Neurospora tyrosinase have been studied by fluorescence spectroscopy. The emission spectra observed for oxy-, deoxy-, met- and apo-tyrosinase and the Co2+-substituted form are indicative of a protein containing buried tryptophan residues. By using acrylamide and iodide, part of the emission is quenched, indicating heterogeneity in the tryptophan environment. Upon binding of Cu2+ or Co2+ to apo-tyrosinase, a marked decrease of the tryptophan quantum yield is observed. A further decrease in emission intensity results from the binding of molecular O2 to the deoxy form. The fluorescent probe 8-anilinonaphthalene-1-sulphonate binds to tyrosinase only when the metal ions are removed. Reconstitution of apo-tyrosinase with Cu2+ completely displaces the probe, suggesting that 8-anilinonaphthalene-1-sulphonate binds to apo-tyrosinase at the active site. The fluorescence properties of Neurospora tyrosinase are compared with those of haemocyanin.     

8-Anilinonaphthalene-1-sulphonate interaction with whole and disrupted mitochondria: a re-evaluation of the use of double-reciprocal plots in the derivation of binding parameters for fluorescent probes binding to mitochondrial membranes.

It is shown that 8-anilinonaphthalene-1-sulphonate is a permeant anion of whole mitochondrial membranes. It is also shown experimentally and algebraically that plots of reciprocal fluorescence against reciprocal membrane concentration, at a fixed 8-anilino-naphthalene-1-sulphonate concentration, are straight lines even when more than one binding site is involved.     

Iron-dependent binding of 8-anilinonaphthalene-1-sulphonate by both lactoferrin and transferrin.

Fluorescence of 8-anilinonaphthalene-1-sulphonate is enhanced by both lactoferrin and transferrin. The enhancement is relatively low between pH 4 and 8, and high at below pH 4. At physiological pH the fluorescence enhancement is higher with the iron-deprived than with the iron-saturated proteins. Binding of iron by lactoferrin is associated with lowering affinity for the dye.     

The binding of 8-anilinonaphthalene-1-sulphonate to a fixed concentration of unenergised and succinate-energised submitochondrial particles

The effect of the approximately hyperbolic relationship between fluorochrome concentration and light absorbed on the interpretation of data for the binding of 8-anilinonaphthalene-1-sulphonate to unenergised and succinate-energised submitochondrial particles has been investigated. If this is taken into account plots of fluorescence against fluorescence x [fluorochrome]-1 do not tend towards a maximum fluorescence value. The significance of this findings is discussed.     

A study on the membrane potential and pH gradient in chromatophores and intact cells of photosynthetic bacteria

Generation of membrane potential (Δψ) and transmembrane pH difference (ΔpH) was studied in PP_i-energized chromatophores of Rhodospirillum rubrum by means of measurements of carotenoid and bacteriochlorophyll absorption changes, atebrin and 8-anilinonaphthalene-1-sulphonate fluorescence responses, and phenyldicarbaundecaborane transport.The data obtained are consistent with the suggestion that carotenoid, bacteriochlorophyll and phenyldicarbaundecaborane responses are indicators of Δψ, while an atebrin response is an indicator of ΔpH. The fluorescence of 8-anilinonaphthalene-1-sulphonate is affected both by Δψ and ΔpH.     

Cell wall composition of ethambutol susceptible and resistant strains of Mycobacterium smegmatis ATCC 607

Changes in the cell wall that accompany acquisition of ethambutol (EMB) resistance ina single step mutant of Mycobacterium smegmatis ATCC 607 were analysed. Quantitative changes were seen in the chemical constituents of the cell wall of resistant cultures in comparison with EMB-susceptible M. smegmatis . Alterations in the binding of 1-anilinonaphthalene-8-sulphonate (ANS) were suggestive of structural changes in the cell surface.     

1-Anilinonaphthalene-8-sulphonate, a fluorescent conformational probe for glutamate dehydrogenase

1. The interaction of 1-anilinonaphthalene-8-sulphonate with ox liver glutamate dehydrogenase was examined. 2. The fluorescence of the dye is enhanced 100-fold on binding. 3. A further enhancement is observed when NADH and GTP are added to the enzyme. 4. By using this property of the dye to measure conformational equilibria in the enzyme the effects of coenzyme, inhibitors, enzyme concentration, ionic strength and pH on the allosteric transitions were studied. 5. GTP and NADH interact with the enzyme in a heterotropic manner. 6. The rate of the structural transition brought about by GTP and NADH is biphasic with half-lives of 34 and 200msec. 7. The relation of these observations to regulatory mechanisms is discussed.     

Physical and binding properties of large fragments of human serum albumin.

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.     

Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M_r 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M_r 52000 (PLAT Y₁) and M_r 45000 (PLAT Y₂) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y₂ had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y₁- and PLAT Y₂-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y₂ accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y₁ and PLAT Y₂ fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y₁ and PLAT Y₂ for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y₁ and PLAT Y₂. The major glutathione S-transferase from the PLAT Y₂ fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y₂-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.     

The interactions of Rose Bengal and other aromatic anionic dyes with mannitol-1-phosphate dehydrogenase fromEscherichia coli

The interaction of Rose Bengal with mannitol-1-phosphate dehydrogenase has been investigated. Binding of this aromatic anionic dye causes a quenching of the protein fluorescence and various changes in the spectral properties of the dye. As is the case with other dehydrogenases, the titration of the enzyme with Rose Bengal, monitoring enhancement in the dye fluorescence at 590 nm, or quenching of the protein fluorescence, can be described by a simple binding model: one dye binding site per enzyme subunit with a dissociation constant of ～2 µM. However, kinetic studies indicate a more complex scheme, since Rose Bengal induces a biphasic time-dependent inhibition of the enzyme. The first phase is over in 1–5 min and is partially reversible, while the second phase is essentially irreversible and continues beyond 1 h. The dyes 8-anilino-1-naphthalene sulfonate and 2-p-toluidinylnaphthalene-6-sulfonate also cause biphasic time-dependent inhibitions of the enzyme. Only mannitol-1-phosphate, and fructose-6-phosphate in the presence of NAD⁺, show high levels of protection against these inhibitory processes. The different effects of coenzymes and substrates on the dye-induced inhibitions support earlier observations from fluorescence studies (preceding paper). A binding scheme describing the interactions of Rose Bengal with the enzyme that is consistent with the experimental results is presented.     

The effect of methemoglobin and ferricytochrome c on the lipid bilayer structure

The effect of ferricytochrome c and methemoglobin on the model phospholipid membrane structure has been investigated using fluorescent probes: 1-anilinonaphthalene-8-sulphonate, 4-dimethylaminochalcone and 3-methoxybenzanthrone. Ferricytochrome c and methemoglobin is found to cause the disordering of the phospholipid bilayer surface region.     

Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments

Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T opt = 85 degrees C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.     

Effect of calcium ions on plasma membrane structure

The structure of thymocyte's plasma membranes was studied as affected by calcium ions (0-1.126 mM). The fluorescence intensity of 1-anilinonaphthalene-8-sulphonate, the epimerization degree of pyrene and fluorescence anisotropy of membrane proteins were studied. The change of electrochemical properties of membranes, conformation of membrane proteins and lipid fluidity has been shown.     

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 to 460-470nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements.     

Ligand-binding properties of proalbumin Christchurch.

Proalbumin Christchurch, a circulating variant of human serum albumin, is secreted from the liver without cleavage of the hexapeptide situated at the N-terminal end of the peptide chain of proalbumin. We compared ligand-binding properties of proalbumin Christchurch and of normal albumin A from the same individual in order to test the effect of the presence of the hexapeptide. The two albumin forms exhibited similar affinities for palmitate, bilirubin, 8-anilinonaphthalene-1-sulphonate and Bromocresol Green. The patterns of endogenous fatty acids bound to the two forms of albumin were slightly different, although the differences were probably not of physiological significance. From these studies it would appear that the propeptide of proalbumin does not alter the protein conformation in such a way as to alter binding sites for organic anions.     

The interaction of triethyltin with a component of guinea-pig liver supernatant. Evidence for histidine in the binding sites

A protein fraction was isolated from guinea-pig liver that binds triethyltin with an affinity of approx. 2x10(6)m(-1) at pH8.0. It was shown that the protein responsible for binding 70% of the triethyltin found in guinea-pig liver after injection of radioactively labelled triethyltin is at most a few per cent of the total liver protein. Evidence is presented from the kinetics of loss of binding and loss of certain amino acids on photo-oxidation with either Methylene Blue or Rose Bengal that each binding site consists of two histidine residues.     

Ligand-binding studies with a 23 kDa protein purified from bovine brain cytosol

Ligand-binding studies were performed with a basic 23 kDa protein purified from bovine brain cytosol. By equilibrium dialysis experiments bromosulfophthalein, dehydroepiandrosterone sulfate and oestradiol-17 beta were demonstrated to bind to the protein with association constants of 1 X 10(6), 1 X 10(4) and 1 X 10(3) l/mol, respectively. Indocyanine green, Evans blue and Rose Bengal were not bound. The protein was further characterized as a phosphatidylethanolamine-binding protein, while phospholipid transfer assays proved negative. The so far investigated binding characteristics of the 23 kDa cytosolic protein, together with previously demonstrated sequence homologies with other known cytosolic proteins, suggest its involvement in lipid metabolism.     

Study on the inhibition of testicular polyadenylate polymerase stimulated by manganese]

Many different types of naturally occurring substances and of drugs were examined as potential inhibitors of a Mn++-stimulated and poly(A)-primed polyadenylate polymerase that was partially purified from rat testis. By far the most active inhibitors were anionic dyes that were either of the diazo naphthyl sulfonate category, such as Evans Blue, or were halogenated derivatives of fluorescein, like Rose Bengal. The characteristics of these in vitro inhibitions, which did not involve any photo-sensitized reactions, were examined in considerable detail.     

Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A.

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we examined the interactions of bromosulphophthalein, oestrone sulphate, haem and bilirubin with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylchone/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. In all four cases, saturation effects were observed. Values of Vmax (v = mol of compound bound/mol of lipid phosphorus) at 25 degrees C were: for bromosulphophthalein, approximately 0.1; for oestrone sulphate, approximately 0.25; for haem, approximately 0.25 (all at pH 7.4); and for bilirubin 0.1--0.2 (at pH 8.2). 3. Limiting values of v/c (c = unbound concentration) as v leads to 0 at 25 degrees C and pH 7.4 are: for bromosulphophthalein, 6.25 x 10(4) litre-mol-1; for oestrone sulphate, 7.8 x 10(2) litre-mol-1; for haem, 4.5 x 10(5) litre-mol-1; and for bilirubin, approximately 1.2 x 10(4) litre-mol-1. For haem the result depends on the assumption that only the monomeric form binds to the lipid. 4. The binding of each compound was decreased by cholesterol; bromosulphophthalein and oestrone sulphate were affected more than haem and bilirubin. 5. Bromosulphophthalein at saturating concentration decreased the limiting values of v/c of the other three compounds by approximately one order of magnitude. 6. By assuming that the interactions with egg phosphatidylcholine resemble those with the phospholipid components of mammalian intracellular membranes the binding data for phosphyatidylcholine, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 92, 51, 98 and 47% of the total bromosulphophthalein, oestrone sulphate, haem and bilirubin respectively may be membrane-bound.     

The effect of ATP on the cell membrane structure]

ATP influence on the structure of plasma membranes thymocytes of cattle was studied. Fluorescence anisotropy of tryptophan residues of membrane proteins, fluorescence anisotropy of 3-methoxybenzanetron and fluorescence intensity of 1-anilinonaphthalene-8-sulphonate were determined. Changes of tryptophan fluorescence anisotropy and of ANS fluorescence intensity were established. It is supposed that the observed changes are connected with the change of membrane proteins structure and plasma membrane charge.