A modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buffer (4.7 mmhos at 25 degrees C) high speed separations (10-12 min analysis time at 800 V/cm) could only be elicited in 20 microm internal diameter (ID) capillaries, at the expense of sensitivity. In the present report, we optimized the background electrolyte as follows: 40 mM aspartic acid (pH=pI=2.77) in the presence of 7 M urea and 0.5% short-chain hydroxyethylcellulose (Mn 27000 Da; apparent pH 3.9 in 7 M urea). As an alternative recipe, the same isoelectric buffer can be supplemented with a mixed organic solvent composed of 4 M urea and 20% acetonitrile (apparent pH 3.66). Due to the much lower conductivity (0.7 mmhos), separations can be carried out at 1000 V/cm in only 10 min, but in larger bore capillaries (50 microm ID), ensuring a five-times higher sensitivity. The gliadin patterns thus obtained are species-specific and allow easy identification of all cultivars tested of both durum and bread wheat. No adsorption of proteins to the silica wall seems to occur and high reproducibility in peak areas and transit times is obtained.
A biologically active spinach ferredoxin was reconstituted from the apoprotein by incubation with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, reduced lipoate and ferric ammonium citrate. Analytical and spectroscopical features of the reconstituted ferredoxin were identical to those of the native one; yield of the reconstitution reaction was 80 7". Yields and kinetic parameters of the enzymic and chemical reconstitution were also compared. The higher efficiency of the enzymic system is ascribed to a productive interaction between rhodanese and apoferredoxin favouring the process of cluster build-up and insertion. The physiological relevance of this synthetic activity is discussed.Important progress has been made over the last few years in the field of iron-sulfur proteins. However, little still is known about the assembly of iron-sulfur clusters within the cell and their insertion into the various apoproteins.Attempts to reconstitute several iron-sulfur proteins by chemical methods have been more or less successful [1,2]. Because of the toxic nature of the reagents employed [3] and the non-specific chemical reactions [4], the physiological relevance of such models is doubtful.Evidence was also reported on the possible involvement of sulfurtransferascs, an ubiquitous class of enzymes [5,6], in the biosynthesis of iron-sulfur clusters [7,8]. 3-Mercaptopyruvate sulfurtransferase activity was found in both mitochondria and cytosol, but its involvement in the formation of the iron-sulfur cluster of adrenodoxin requires cysteine transaminase activity which is present almost only in the soluble fraction [7]. Rhodanese (thiosulfate-cyanide sulfurtransferase) activity was specifically found in mitochondrial fraction [9] and in chloroplasts where its activity appears to be related to active sulfur metabolism [lo].Rhodanese can restore chemical and functional properties, which have becn lost as a consequence of the alteration of the iron-sulfur cluster(s), in some iron-sulfur proteins such as mitochondrial succinate dehydrogenase [I 11 and NADH dehydrogenase [I21 as well as in the ferredoxins from either Clostridiumpasteurianum or spinach chloroplasts [I 31. A small amount of sulfur from radioactive thiosulfate was found inserted in the iron-sulfur protein as acid-labile [35S]sulfide [14,15]. The reducing equivalents which are necessary for the reduction of the sulfane sulfur of thiosulfate to sulfide were derived from the oxidation of sulfhydryl groups either on the iron-sulfur protein or on the sulfurtransferase itself [14 -161. In the latter case, a strong inactivation of rhodanese occurred. These results proved that rhodanese exerts a protective action on iron-sulfur proteins but they still do not provide direct evidence for an involvement of the sulfurtransferase in the exn o w synthesis of iron-sulfur clusters.Rhodanese is able to produce inorganic sulfide in the presence of its putative biological substrate, thiosulfatc, and of suitable dithiols, such as dihydrolipoate [I 71 o...
The interaction of the sulfurtransferase rhodanese (EC 2.8.1 .l) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied.Succinate Sulfur release from rhodanese appears to depend on the presence of -SH groups in the acceptor protein.Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron-sulfur absorption of the flavoprotein.The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation.The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.Recent reports indicate that sulfurtransferases may be involved in sulfur transfer to iron-sulfur proteins. Taniguchi and Kimura [2] found that adrenodoxin could be reconstituted upon treatment of its apoprotein with 3-mercaptopyruvate and 3-mercaptopyruvate sulfurtransferase ; in another system, rhodanese (an enzyme that transfers sulfane sulfur) and thiosulfate (its substrate) substitute for inorganic sulfide in the reaction medium for restoring the iron-sulfur center of ferredoxin [3]. Details concerning the role of the sulfurtransferase enzyme in these reactions are not yet known.We have studied the interaction of rhodanese with the iron-sulfur flavoprotein succinate dehydrogenase. This paper is VII in a series on succinate dehydrogenase. The previous paper appeared elsewhere [I].
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