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Sedimentation analysis and light-scattering studies indicate that the aggregation state of the pyruvate dehydrogenase complex of Azotobucter vinelundii in 50 mM potassium phosphate (pH 7.0) can be described in terms of a monomer-dimer equilibrium with a dissociation constant of 6.8 pM. The apparent molecular mass of the monomeric particle is 750000 -850000 Da.The equilibrium is shifted to the monomeric species when pressure is applied on the system. Pressure-jump experiments yielded a relaxation time of about 70 ms. In the presence of 3 % poly(ethy1ene glycol) 6000 and 10 mM MgCI2, further association takes place to a system that can be described in terms of dimer-tetramer-octamer equilibria. Upon applying a pressure of 80 MPa to this system these equilibria are shifted to the dimeric state but some monomer formation cannot be excluded. Release of pressure shows that the relaxation time of the dimertetramer equilibrium is less than 5 s, that of tetramer-octamer equilibrium is of the order of minutes. The isolated E2 component has a molecular mass of 2000000k 100000 Da; and thus consists of about 30 E2 peptide chains. Electron micrographs are similar to those of the E2 component of the Escherichiu coli complex, which were interpreted as cubic structures with an octagonal symmetry. Upon addition of El to the pure E2 component, changes in the assembly occur and mixtures of large (E. coli-like, 22 -45 S) and small ( A . uinelundii-like, 11 -I8 S) subcomplexes are obtained. The two forms of the subcomplexes are in slow equilibrium (relaxation time 10 -30 min).It is proposed that the E2 tetramer of the intact pyruvate dehydrogenase complex of A . vinelundii is represented by the corner structures of the isolated E2 component.Pyruvate dehydrogenase complexes (PDC) catalyse the oxidative decarboxylation of pyruvate to yield acetyl-coenzyme A and NADH. Three enzymes participate in this reaction: pyruvate dehydrogenase (El), lipoate acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). These enzymes form large assemblies of non-covalently associated polypeptide chains [l 1. The well-studied PDC of Escherichiu coli has a sedimentation coefficient of 53 -60 S and estimations of its molecular mass range over 3.75-6.1 MDa [2 -51. PDCs from gram-positive bacteria and eukaryotes are even larger [6 -81. The PDC from Azotobucter vinelundii is the smallest complex known : its sedimentation coefficient was determined to be 19 S and from light-scattering measurements a molecular mass of l -l .2 MDa was calculated [9]. The A . vinelundii PDC preparations that were used in these experiments had a low specific activity (6 U/mg protein) and contained the so-called fourth component [9,10]. We have therefore repeated these measurements with the pure threecomponent, high-specific-activity preparations (1 5 -19 U/mg protein) that were obtained by a revised purification procedure [ll].Abbreviations. PDC, pyruvate dehydrogenase complex; PEG 6000, poly(ethy1ene glycol) 6000; SDS, sodium dodecyl sulfate.Enzymes. Pyruvate dehydrogenase...

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Hydrodynamic radii from inelastic light-scattering experiments and radii of gyration from Zimm plots give an indication of the change of average casein micelle size when the pH is changed. Combination of the results of both types of measurements gives information on changes in the micelle protein matrix, i.e. changes in the voluminosity.The voluminosity was also determined by the pellet method and by electron microscopy which also provided comparative data on size parameters.Fermentation of milk to obtain a variety of consumable acid gels and fluids is an important process in the dairy industry. To understand the changes in milk that bring about the eventual physical properties of the products, detailed knowledge on a molecular level is needed of what happens to casein micelles when milk is acidified. This study combines the results of an intrinsically number-average technique, electron microscopy, with weight and Z-average techniques offered by light scattering. In this way the whole casein micelle size distribution can be studied for acidified milk samples.Data were obtained for a slightly pasteurized milk which is used extensively in cheese-making in the Netherlands. MATERIALS AND METHODS VoluminositySkim milk was cooled to 4 °C and the pH adjusted to the desired value; the milk was stored overnight at 4 °C. In the morning the milk was heated to 30 °C and kept at that temperature. The pH was checked and readjusted if necessary. After 1 h the pH was checked again. The samples were spun at 29000 rev/min (88000 g) for 2 h in a Beckman preparative ultracentrifuge at 30 °C using the 30 rotor, which had been preheated to 30 °C. The pellet was weighed before (w^) and after (w 2 ) freeze drying. The freeze-dried pellet was ground in a mortar and the nitrogen content of the powder (N) was determined. The voluminosity was calculated from: _ ( K -w 2 ) + w 2 [0-55 + (0-74-0-55) Nx 6-35/100]} V~ iv 2 Nx 6-35/100