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PubMed1992

Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation.

The dimerization of phospholipase A2 (PLPA2) from Naja naja naja (Pakistani cobra) and Crotalus atrox (Western Diamondback rattlesnake) has been studied from pH 2.5 to 11 at 20 degrees C in 1 mM CaCl2, 0.21 M ionic strength. For the C. atrox enzyme, it was found necessary to use a combination of sedimentation equilibrium and fluorescence yield data to analyze the association. Sedimentation equilibrium in the analytical ultracentrifuge sufficed for the study of the N. naja PLPA2. In the region of enzymatic activity at pH 8, the dimerization association constants found were k2 = 2.8 X 10(6) L/mol and k2 = 6.9 X 10(4) L/mol for the C. atrox and N. naja enzymes, respectively. Analytical linked functions are presented which describe the data. Because the associations are linked to Ca2+ as well as the hydrogen ion, no attempt was made to interpret the ionization of residues in terms of the molecular structure. Active-enzyme sedimentation velocity experiments have been used to study the relation between enzymatic activity and association for both the C. atrox and N. naja enzymes. The substrate 1,2-dibutyryl-sn-glycero-3-phosphocholine (diC4PC) did not dissociate the C. atrox PLPA2. The substrate 1,2-dihexanoyl-sn-glycero-3-phosphocholine (diC6PC) at 7.5 mM dissociated the C. atrox PLPA2 when monitored either as the active enzyme or as the Sr2+-inhibited enzyme. At low enzyme concentrations, 40 mM diC4PC had no effect on N. naja PLPA2 dimerization. However, the sedimentation coefficients observed at enzyme concentrations above 0.2 mg/mL in active-enzyme sedimentation velocity experiments were larger than the values predicted from the thermodynamic studies. Sedimentation coefficients observed for the N. naja PLPA2 acting on diC6PC were larger than those of the monomeric protein, which was the form layered on this substrate. The dissociation of the C. atrox PLPA2 effected by diC6PC was analyzed by the thermodynamics of association and the kinetic Michaelis constant. The analysis suffices to account for the observed sedimentation coefficient. The sedimentation behavior of the N. naja PLPA2 acting on diC6PC substrate was analyzed in terms of a protein-lipid complex. With this model, 68 +/- 16 phospholipid molecules per protein monomer were determined. It is proposed that this enzyme has two micelle nucleation sites per monomer. These putative sites promote micelle formation of the substrate on the enzyme below the critical micelle concentration of the lipid alone.

Animals↗
PubMed2004

Abnormal micellar growth in sugar-based and ethoxylated nonionic surfactants and their mixtures in dilute regimes using analytical ultracentrifugation.

To develop structure-property relationships for surfactants that control their adsorption, solubilization, and micellization behavior in mixed systems and to develop predictive models based on such relationships, it is necessary to acquire quantitative information on various species present in these complex systems. The analytical ultracentrifugation technique is selected for the first time to characterize the species present in mixed micellar solutions due to its powerful ability to separate particles on the basis of their size and shape. Two nonionic surfactants, n-dodecyl-beta-D-maltoside (DM) and nonyl phenol ethoxylated decyl ether (NP-10), and their 1:1 molar ratio mixture were investigated in this study. Micelles of the nonionic surfactants and their mixture are asymmetrical in shape at the critical micelle concentration (cmc). Interestingly, unlike ionic surfactants, the micellar growths of the nonionic surfactants were found to occur at concentrations immediately above the cmc. The results from both sedimentation velocity and sedimentation equilibrium experiments suggest coexistence of two types of micelles in nonyl phenol ethoxylated decyl ether solutions and in its mixture with n-dodecyl-beta-D-maltoside, while only one micellar species is present in n-dodecyl-beta-D-maltoside solutions. Type 1 micelles were primary micelles at the cmc, while type 2 micelles were elongated micelles. The differences in the micellar shapes of n-dodecyl-beta-D-maltoside and nonyl phenol ethoxylated decyl ether are attributed to packing parameters detected by their molecular structures.

Journal Article↗
PubMed1974

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by density-gradient ultracentrifugation.

1. Equilibrium density-gradient ultracentrifugation in caesium salts was used in two stages in the isolation and subfractionation of the glycoprotein component from a human ovarian-cyst fluid. The eight main subfractions thus obtained were the subject of detailed physicochemical characterization. 2. The fractions were unimodal in buoyant-density distribution, but had discrete rho(0) values ranging from 1.31 to 1.35. 3. Weight-average molecular weights and sedimentation coefficients decreased regularly with decreasing density of the fraction, whereas the partial specific volumes and selective solvation parameters increased. The latter behaviour correlates well with the increasing peptide content of the lighter fractions. 4. The fractions exhibited a range of analytical composition, although all were within the limits previously observed for blood-group substances of Le(a) specificity. All fractions had approximately equal Le(a) activity. The peptide content varied systematically from 7% for the densest fraction to 15% for the lightest, but the relative distributions of the amino acids remained essentially constant throughout the series. In particular, serine plus threonine plus proline made up about 50% of the peptide content of all the fractions. Fucose, galactose and N-acetylglucosamine contents decreased with increasing peptide content of the fractions, but N-acetylgalactosamine and sialic acid exhibited the opposite trend. Molar ratios of N-acetylgalactosamine to the sum of serine and threonine remained essentially constant at 0.8-0.9, implying a high degree of glycosylation of all the molecules, but the ratio of N-acetylglucosamine to N-acetylgalactosamine decreased steadily with increasing peptide content, suggesting the presence of oligosaccharide side chains of various lengths. The results are discussed in terms of the accepted structure of glycoprotein molecules. 5. Experiments on the glycoproteins extracted with phenol from the same cyst fluid have confirmed that equilibrium centrifugation in caesium salts does not remove any non-covalently bound protein nor cause any changes in the tertiary structures of these glycoprotein molecules.

Acetylgalactosamine↗
PubMed2003

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation.

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Cyclotides↗
PubMed1977

Separation of subclasses of human serum high density lipoproteins by zonal ultracentrifugation.

An improved one-step method for the preparative separation of three subfractions of high-density lipoproteins from normal human serum has been developed. It employs the method of rate zonal ultracentrifugation in a z-60 rotor using a discontinuous NaBr gradient in the density range of 1.0-1.4. The density gradients were monitored directly by a flow-through density meter allowing the direct read-out of the actual densities in the process of filling and emptying the rotor. The separation of the three density fractions from 5 to 15 ml serum was achieved during a single 12 hours run at 59.000 rpm. The three fractions showed characteristically different patterns on polyacrylamide gel electrophoresis and differences in their lipid and protein composition.

Centrifugation, Zonal↗
PubMed1969

The effect of ultracentrifugation on fine structure and alpha-amylase production in barley aleurone cells.

Ultracentrifugation of barley aleurone cells results in the stratification of organelles thus allowing for a quantitation of those organelles. Gibberellic acid (GA(3))-stimulated alpha-amylase production in stratified cells is reduced by centrifugation at gravitational forces greater than 40,000g. Forces below 30,000g do not affect GA(3)-stimulated alpha-amylase production although stratification of organelles occurs at these forces. The ability of centrifuged cells to respond maximally to GA(3) by producing alpha-amylase is related to the degree of redistribution of organelles within these cells. Thus, recovery of cells from centrifugation at forces below 30,000g is rapid, while recovery from forces above 40,000g is slow.

Journal Article↗
PubMed1982

An attempt to localize the vaccinating power of Klebsiella pneumoniae ribosomal preparations using saccharose-gradient ultracentrifugation.

The study of the vaccinating power of ribosomal vaccines against Klebsiella pneumoniae led us to define the chemical nature which supports this protective activity. We tried to separate this support and the ribosomes by sucrose gradient ultracentrifugation. We isolated high protective membrane vesicles by this technique applied to salt-washed ribosomal preparations. When the ribosomal preparations were exposed to SDS, the protective activity was conserved all along the gradient, with no correlation with the ribosome concentration. The addition of bovine serum albumin to the ribosomal preparation focused the protective activity on the ribosomal peak. No correlation was observed between the response to capsular polysaccharide and the vaccinating power of the fractions.

Ammonium Chloride↗
PubMed1985

Complexed autoantibodies in patients with juvenile connective tissue diseases, isolated by rate-zonal ultracentrifugation.

Selected sera from one patient with systemic lupus erythematosus, two with mixed connective tissue disease, one with dermatomyositis, one with progressive systemic sclerosis and one with juvenile rheumatoid arthritis were investigated for autoantibodies after fractionation by computerized rate-zonal ultracentrifugation. Anti-Smith antibodies sedimented in an area from 6-11 S and anti-ribonucleoprotein from 6-13 S. IgG anti-IgG and IgG antinuclear antibodies (ANA) were present in free or complexed form in the 6-13 S area. IgM ANA occurred as 7 S IgM in patients with systemic lupus erythematosus and mixed connective tissue disease, whereas IgM ANA sedimented in the 19 S area in patients with dermatomyositis and progressive systemic sclerosis. Complexes containing IgG anti-IgG and ANA, positioned in the 6-13 S area are likely to play a significant role in the pathogenesis of systemic lupus erythematosus and mixed connective tissue disease.

Adolescent↗
PubMed1962

Separation of cell components in the zonal ultracentrifuge.

Zonal separation of particles in density gradients contained in hollow or tubeless rotors has been explored with the aim of developing a preparative counterpart of the analytical ultracentrifuge. A rotor with a capacity of 1625 milliliters has been tested up to 22,500 revolutions per minute, with sucrose density gradients used. Excellent separation of subcellular particles has been achieved as well as partial separation of the albumin and globulin peaks of serum.

Cell Biology↗
PubMed1968

Use of zonal ultracentrifuge systems for biophysical studies of rhinoviruses.

This paper reports the use of zonal ultracentrifuge techniques to conduct biophysical studies of rhinoviruses grown with WI-38 cells. Good clean-out of infectivity from rhinovirus harvests was obtained with the continuous-flow B-V and B-IX rotors. Use of the B-V rotor resulted in the successful concentration of rhinovirus infectivity and antigenicity. Additional purification was achieved by the combined use of continuous-flow centrifugation and isopycnic banding procedures. Two particle sizes were found to be associated with the virus-infected cell harvests. The infectious 22-nm particle banded in density ranges of 1.38 to 1.40 g/cm(3) in CsCl and 1.26 to 1.27 g/cm(3) in potassium citrate. The 8.0 nm capsomere was composed of 2.0 nm subunits and banded with a density of protein at 1.28 g/cm(3) in CsCl. Equivalent sedimentation coefficients of 155 or 185, depending on particle density in sucrose, were calculated from rate zonal experiments by use of the B-IV zonal rotor.

Centrifugation, Density Gradient↗
PubMed1995

Determination of total and herpes simplex virus specific monomeric and dimeric IgA in serum and cerebrospinal fluid by ultracentrifugation.

An improved method is described for differentiating between monomeric and dimeric total and herpes simplex virus (HSV) specific IgA by ultracentrifugation in sucrose gradient, using recovery and quantitative analysis of the fractions obtained. Calculation of monomeric and dimeric IgA was based on IgG as an internal standard. Intrathecally produced monomeric and dimeric IgA were judged by calculating IgA indices for each form. A new type of formula indicating relative over-production of dimeric compared with monomeric IgG (IgA dimeric-monomeric index) is suggested. The method was applied to serum and cerebrospinal fluid (CSF) from three patients with HSV encephalitis. The index for monomeric as well as dimeric IgA was high during the acute phase of the disease, indicating intrathecal synthesis of both molecular forms. One year after onset, there was no detectable HSV-specific IgA in CSF: both molecular forms, however, remained in serum. The amount of dimeric compared with monomeric IgA was high during the acute phase, and subsequently decreased after successful treatment. A new finding was the detection of HSV-specific IgA heavier than dimeric IgA in serum one year after onset of the disease. These components may be tetrameric IgA, or immune complexes containing IgA.

Antibodies, Viral↗
PubMed1977

Ultracentrifugal characterization of cytosol-binding proteins for thyroxine and triiodothyronine in human liver.

Sucrose density gradient ultracentrifugation was performed on human liver cytosol containing double tracers of 131I-thyroxine(T4) and 125I-triiodothyronine(T3) together with or without large amount of stable hormones. Tracer T4 bound to the cytosol indicated 2 peaks of 8.3S and 4.7S, both of which were displaced with stable T4, but not with T3. Maximal binding capacity of the 8.3S component averaged 43.2 microgram/100ml of 33% cytosol. Tracer T3 in the cytosol showed a single peak of 4.2S which was displaced with stable T3 and also with T4. The results indicate thtat the human liver cytosol contains limited-capacity binding proteins: the one specific for T4, and the other relatively specific for T3.

Carrier Proteins↗
PubMed2003

Rapid separation of LDL subclasses by iodixanol gradient ultracentrifugation.

BACKGROUND: A predominance of small, dense LDL (sdLDL) confers in excess of a threefold increase in coronary heart disease (CHD) risk. The conventional method for the detection of sdLDL, salt density gradient ultracentrifugation (DGUC) has been superseded by more rapid techniques. This report presents novel methodology for the separation of sdLDL by a combination of iodixanol density gradient centrifugation and digital photography. METHODS: LDL subclasses were separated in 3 h from prestained plasma on a self-forming density gradient of iodixanol. LDL subclass profiles were generated by digital photography and gel-scan software. Plasma samples from 106 normo- and dyslipidemic individuals were used to optimize the gradient for the resolution of LDL heterogeneity. A subgroup of 47 LDL profiles were then compared with LDL subclasses separated by salt DGUC. RESULTS: The peak density of the predominant LDL band correlated significantly with the relative abundance (as a percentage) of sdLDL as resolved by salt DGUC (P <0.001). As shown previously, LDL isolated at a lighter density in iodixanol compared with salt gradients. A predominance of sdLDL corresponded to a peak density on iodixanol of 1.028 kg/L. This density and the area under the LDL profile lying above this density were sensitive and specific markers for the prediction of a predominance of sdLDL (P <0.001) and showed predictable associations with plasma triglycerides (r = 0.59; P <0.001) and HDL (r = -0.4; P <0.001). CONCLUSIONS: This simple method for the detection of sdLDL can differentiate a predominance of sdLDL, is highly reproducible, and can be used preparatively to isolate sdLDL.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation.

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.

Animals↗