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We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS-agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/l) of the individual fractions for ristocetin-dependent binding to GP Ib and thrombin-induced binding to GP IIb/IIIa. Direct binding studies of the 125I-vWF pool gave mean Kd values of 2.02 +/- 0.05 x 10(-8) for GP Ib and 1.15 +/- 0.02 x 10(-8) for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3-10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin- and thrombin-induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.

A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelaizdii on a 2.3-kb SphI fragment. This fragment was identified by its hybridization to a PCR product obtained by amplification of genomic DNA using degenerate primers encoding the N-terminal sequence of rhodanese purified from A. vinelandii. The sequence of a 1.2-kb region revealed an 813-bp open reading frame that encoded a polypeptide of 271 amino acids, the N-terminal sequence of which was identical to that of A. vinelandii rhodanese. In a search of database entries, eukaryotic rhodaneses and rhodanese-like proteins from bacteria gave the highest scores of identity (27-30%) with the predicted product of the 813-bp open reading frame. A. vinelandii RhdA shows less sequence similarity to vertebrate rhodaneses than it does to prokaryotic rhodanese-like proteins which did not show typical rhodanese activity. Basic residues thought to be catalytically important in bovine rhodanese are not conserved in A. vinelandii rhodanese. The sequence similarity between the two structurally similar domains of rhodanese is more pronounced for the A. vinelandii enzyme than the bovine enzyme, and supports the hypothesis that the complete structure was originally generated by gene duplication. When rhdA was overexpressed in Escherichia coli, rhodanese represented 30% of total cell protein and thiosu1fate:cyanide sulfurtransferase activity increased > 600 fold in cellfree extracts. A. vinelandii rhdA insertion/deletion mutants had no discernible phenotype distinct from the wild-type strain with respect to growth on various sulfur sources or nitrogenase activity. Mutants retained 20 % of wild-type rhodanese thiosulfate :cyanide sulfurtransferase activity suggesting the presence of redundant sulfurtransferase enzymes in A. vinelandii.

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...

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