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We have recently reported (Journal of Biological Chemistry 261: 15310-15315, 1986) that von Willebrand factor (vWF) possesses a collagen-binding domain localized in a reduced and alkylated tryptic fragment of apparent 52/48 kDa molecular weight extending between residues Val (449) and Lys (728) of the constituent subunit. This proteolytic fragment of vWF also contains a glycoprotein lb-binding domain and a heparin-binding domain. We have now identified a second collagen-binding domain in the Staphylococcus aureus V8 protease-generated fragment I that extends from residue Gly (911) to Glu (1365). The two binding domains exhibit different interaction with collagens of different origin. The reduced and alkylated 52/48 kDa tryptic fragment was a potent inhibitor of vWF binding to equine collagen type I, but had no effect on the binding to bovine collagen type I and III. In contrast, a purified fraction containing the unreduced 52/48 kDa domain inhibited vWF binding to all types of collagen, as did anti-52/48 kDa monoclonal antibodies. Some of these antibodies, however, were more effective in inhibiting binding to equine collagen. On the other hand, fragment I markedly inhibited the binding of vWF to bovine collagen type I and III, but was less effective with equine collagen type I. Direct binding studies using 425j_qabeled fragment I demonstrated that the association constant was 5 to 10 times greater with the bovine collagens than with the equine collagen. The Staphylococcus aureus V8 protease-generated fragment III, which extends from residue Ser (1) to Glu (1365) and contains both collagen-binding domains, was the most potent inhibitor of vWF binding to all types of collagen tested. Thus, vWF has at least two collagen-binding domains. Native conformation appears to be necessary for binding of the 52/48 kDa domain to bovine collagen type I and III, but not to the equine collagen type I tested. The two domains appear to function concurrently in mediating vWF binding to collagen.

The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaena cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective electron donors to nitrogenase in crude extracts whereas hydrogen and pyruvate were not. The K , for acetylene in vivo is ten-fold higher than the K,,, in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2-content (2,20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A . cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubvum, and P. boryanum.The presence of nitrogenase, the enzyme that catalyzes biological fixation of atmospheric nitrogen, has been described in a variety of prokaryotic organisms, including some species of cyanobacteria [I -51. Enzyme activity has invariably been found to be dependent on a source of ATP and reductant, and to be irreversibly inactivated by oxygen. Most of the cyanobacteria known to fix nitrogen belong to filamentous, heterocyst-forming families which are capable of fixing nitrogen under aerobic conditions. The cyanobacterial nitrogenase is thought to be localized in these heterocysts, specialized differentiated cells which have a restricted photosynthetic process that does not evolve oxygen, due to the absence of photosystem I1 components. The thick cell wall structure of heterocysts is postulated to provide protection from oxygen inactivation by serving as an effective barrier to the diffusion of gases [6,7].Nitrogenase activity in vivo is maximal only in the light, indicating dependence on photosystem I but is not directly dependent on photosystem I1 activity [8,9]. Thus, the major pathways for supply of reductant (reduced ferredoxin) probably involve the metab- Enzymes. Nitrogenase or nitrogen: (acceptor) oxidoreductase (EC 1.7.99.2): creatine phosphokinase (EC 2.7.3.2).

Among affected members of a family (B. family) with excessive purine production and gout, activity of phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) is 2.5- to 3.0-fold higher than among normal people. The molecular basis for this increased enzyme activity was studied. Antibody to purified human phosphoribosylpyrophosphate synthetase of erythrocytes was obtained from immunized rabbits. Studies with the IgG fraction of this antiserum show the presence of normal quantities of immunoreactive enzyme, but 2.5- to 3.0-fold higher activity per molecule in affected members of the B. family. In addition, by use of a stain for phosphoribosylpyrophosphate synthetase activity, a difference in electrophoretic mobility was demonstrated on cellulose acetate gel between the partially purified enzyme from normal people and an affected member of the B. family. These studies suggest that the enzyme aberration responsible for purine overproduction and gout in the B. family results from a structurally altered enzyme with increased activity per molecule.

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