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PubMed1969

Ultracentrifuge tudies of histone fractions from calf thymus deoxyribonucleoprotein.

Molecular weights and sedimentation coefficients of four major fractions of calf thymus histones were measured. The minimum molecular weights were determined in concentrated solutions of guanidine hydrochloride. The results indicate that, with the possible exception of fraction F3, the fractions are heterogeneous. Comparisons in 0.1m-sodium chloride suggest that fraction F1 does not aggregate and show that fractions F2(a) and F3 aggregate to form larger complexes than does fraction F2(b). The degree of aggregation of each fraction is independent of pH in the range pH1-7. Detailed studies with fraction F2(b) have confirmed that the change in sedimentation coefficient observed as the sodium chloride concentration of the solution is increased results from increases in the apparent molecular weight of the sedimenting units. It has been found that the molecules of fraction F2(b) are present as single molecules only in sodium chloride solutions of 33mm or less. At these low concentrations the effects of charge greatly increase the concentration dependence of the sedimentation rate; the results can, however, be interpreted by using the theory developed by Alexandrowicz & Daniel (1963) and Daniel & Alexandrowicz (1963).

Animals↗
PubMed1973

Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation.

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na(2)S(2)O(4) at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)-(Fe+Mo-containing protein).

Bacterial Proteins↗
PubMed1982

A simple test for macromolecular heterogeneity in the analytical ultracentrifuge.

A simple check for the presence of heterogeneity in a macromolecular system is proposed, employing comparison of Rayleigh sedimentation-equilibrium patterns for two solutions of the same fringe concentration but differing absolute concentrations. The method is illustrated by application to a bronchial glycoprotein from a cystic-fibrosis patient.

Glycoproteins↗
PubMed2001

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b.

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as well as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding by trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T<==>R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.

Animals↗
PubMed1999

Self-association and domains of interactions of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by analytical ultracentrifugation and NMR experiments in trifluoroethanol/H(2)O mixtures.

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.

Amino Acid Sequence↗
PubMed1955

Effect of blood thrombokinase, as influenced by soy bean trypsin inhibitor, ultracentrifugation, and accessory factors.

1. Crystallized soy bean trypsin inhibitor, at a concentration of 100 microg./ml., suppressed the production of thrombin from a mixture of prothrombin and blood thrombokinase. The experiment was performed in the presence of 0.011 M oxalate, in order to minimize the possibility of participation by accessory factors which require ionic calcium. The results are in accord with the view that thrombokinase is a trypsin-like enzyme. 2. When a solution of blood thrombokinase was centrifuged at 85,000 g for 120 minutes, almost all the activity remained in the supernate. This supernate activated the supernate from a prothrombin solution which had been similarly centrifuged. The activation of prothrombin by thrombokinase can proceed in the absence of material completely sedimentable in 120 minutes at 85,000 g. 3. An "accelerator" reagent was prepared by treating bovine serum with barium carbonate, and then passing the serum through a column of diatomaceous earth. This "accelerator" was used together with prothrombin, blood thrombokinase, Howell's cephalin, and calcium chloride to compose a five-reagent thrombin-producing system. In this system, no thrombin was produced without thrombokinase. On the other hand, thrombin was produced from prothrombin and thrombokinase, even when all the other reagents were omitted. When calcium was omitted, thrombokinase was able to function; but cephalin and the "accelerator" reagent were ineffective. 4. Quantitative tests indicated that the "accelerator" reagent exerted an effect distinct from those of thrombokinase and cephalin. However, it is not certain whether the "accelerator" reagent functioned as an accessory factor, as a potential source of more thrombokinase, or both. In the experiments reported, thrombokinase was primary to, or necessary for, the effect of "accelerator." 5. The effectiveness of thrombokinase was multiplied a hundred times or more, when complemented by calcium, cephalin, and "accelerator" reagent. Ionic calcium was a necessary component of this complementing system. This may help to explain why removal of calcium ions keeps blood fluid, even though thrombokinase, by itself, is little influenced either by calcium ions or by oxalate.

Animals↗