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PubMed2000

Reactions of direct LDL-cholesterol assays with pure LDL fraction and IDL: comparison of three homogeneous methods.

According to the definition of the Lipid Research Clinic's protocol, low-density lipoprotein (LDL) refers to the lipoprotein of density (d)=1.006-1.063 g/ml which contains another atherogenic lipoprotein, IDL (d=1.006-1.019 g/ml). Because metabolic properties are largely different between LDL and IDL, LDL is now defined as the lipoprotein of d=1.019-1.063 g/ml. Recently direct LDL-cholesterol assay kits using novel surfactants (the homogeneous methods) have become commercially available and widely used in Japan. The aim of this study is to examine how three direct LDL-cholesterol assay kits, LDL-EX, Choletest-LDL and Determinor-L LDL, react with pure LDL (d=1. 019-1.063 g/ml) and IDL (1.006-1.019 g/ml) fractions isolated by ultracentrifugation. Thirty-one healthy subjects and one type III dysbetalipoproteinemic patient were enrolled in this study. All homogeneous methods highly correlated with LDL-cholesterol (r=0.95-0. 98), although the values for LDL-EX were closer to the values for ultracentrifugation than were those of the other two methods (95 vs. 86-87%, P<0.0001). Cross-reactivity with IDL was 31, 47 and 64% for LDL-EX, Choletest-LDL, and Determinor-L LDL, respectively. Similar results were obtained in the IDL from a type III dysbetalipoproteinemic patient. These results suggest that LDL-cholesterol measured by LDL-EX better reflects pure LDL fraction with weaker cross-reaction with IDL than other homogeneous methods.

Cholesterol↗

PubMed1997

Direct isolation of labeled low density lipoproteins for the determination of cholesteryl ester transfer protein activity.

The measurement of the activity of cholesteryl ester transfer protein (CETP), is of high clinical interest and this study reports the use of a direct LDL isolation (d-LDL) technique to determine in one step the amount of radiolabeled cholesteryls esters ([3H]-CE) transferred from exogenous HDL3 to LDL, avoiding the conveniences of the usually used ultracentrifugation or precipitation of apo-B containing lipoproteins in the CETP methodologies. The d-LDL technique providing a specific immunoprecipitation of VLDL, IDL and HDL allowed to directly determine the [3H]-CE transferred on LDL (d-[3H]-CE-LDL). Two methodologies were assayed for the CETP activity using either exogenous or endogenous lipoproteins, and the results with the d-LDL technique were compared with those obtained using the ultracentrifugation (u-[3H]-CE-LDL) considered as the reference method. The intra- and inter-assays were similar in both techniques for the two CETP activity assays. Strong positive correlations were established between values obtained with d-[3H]-CE-LDL and u-[3H]-CE-LDL isolation procedures for CETP activities with exogenous or endogenous lipoproteins (r = 0.972; p = 0.0001 and r = 0.965; p = 0.0001 respectively). In conclusion, the d-LDL technique represents an easy and accurate procedure to measure directly, in normotriglyceridemic plasmas, the amount of [3H]-CE transferred from HDL to LDL by the CETP.

Carrier Proteins↗

PubMed1981

Quantification of sinking pre beta lipoprotein in human plasma.

Two low density protein carriers of plasma cholesterol (beta-lipoprotein and sinking pre-beta-lipoprotein -- a genetic variant of beta-lipoprotein with pre-beta electrophoretic mobility) were isolated from human plasma by ultracentrifugation and gel filtration. They were subjected to agarose gel electrophoresis, followed by staining with Sudan Black B, and their concentration was determined by optical densitometry. The staining of the low lipoproteins was proportional to their total cholesterol the low lipoproteins was proportional to their total cholesterol content over a range of 2-50 mg/dl for sinking pre-beta-lipoprotein (SPB) and 50-250 mg/dl for beta-lipoprotein. Mixing experiments of the two purified lipoprotein preparations indicated that the cholesterol in the SPB could be estimated by the formula: SPB cholesterol = TBC x OD of SPB/(OD of SPB + OD of beta cholesterol), where OD is optical density and the TBC was either the total cholesterol content of mixtures of purified beta- and SPB-lipoproteins, or was determined as the total plasma low density lipoprotein cholesterol content measured by ultracentrifugation and polyanionic precipitation of beta- and SPB-lipoproteins from plasma. The SPB cholesterol values calculated by the above formula deviated from the true value (amount added) by a mean of 6% when beta-lipoprotein cholesterol was present at concentrations of 100, 165, and 216 mg/dl. The deviation did not correlate with the concentration of beta-lipoprotein cholesterol. The coefficient of variation for the determination of SPB lipoprotein by the scanning procedure wa 5-10% for SPB cholesterol concentrations of 6-30 mg/dl. SPB concentrations of 1-2 mg/dl cholesterol could be detected, but the method was unsatisfactory for quantification below 4 mg/dl cholesterol. By providing an estimate of the distribution of cholesterol between the subfractions of low density lipoprotein, the method should prove helpful in assessing risk of atherosclerosis.

Cholesterol↗

PubMed1998

Baseline diene conjugation in LDL lipids as a direct measure of in vivo LDL oxidation.

OBJECTIVES: To additionally test validity of the recently developed method (LDL baseline diene conjugation, LDL-BDC) for determination of circulating oxidized LDL. DESIGN AND METHODS: A detailed comparison between the ultracentrifugation and heparin precipitation methods for LDL isolation was performed to test suitability of the fast precipitation method. Validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing LDL-BDC to results obtained by the immunological autoantibody method. RESULTS: BDC values in LDL isolated by heparin precipitation did not differ from those isolated by sequential ultracentrifugation. While highest amount of diene conjugation was found in LDL (40% of that in serum), substantial amounts were also found in VLDL (31%) and HDL (25%). When analyzed in the same samples, assays for the titer of autoantibodies against oxidized LDL and LDL-BDC were found to show good correlation (r = 0.57, p = 0.001, n = 29). CONCLUSIONS: These results, together with thus far conducted studies on clinical applicability of the method, indicate that LDL-BDC is a promising candidate in search for a method for estimation of LDL oxidation in vivo.

Adult↗

PubMed1999

A 6 bp Z-DNA hairpin binds two Z alpha domains from the human RNA editing enzyme ADAR1.

The Z alpha domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Z alpha domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Z alpha and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (> or = 6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

Adenosine Deaminase↗

PubMed1994

Increase of lymphocytes expressing Fc-receptor class III (CD16) by exposure to human seminal plasma.

OBJECTIVE: To characterize conditions for the seminal plasma-induced increase of lymphocytes that express immunoglobulin G-Fc receptor class III (Fc gamma RIII). DESIGN: Peripheral blood lymphocytes were incubated in diluted seminal plasma or control media. Cells expressing Fc gamma RIII were quantified by using flow cytometry and fluorochrome-labeled monoclonal antibodies specific for Fc gamma RIII. The influence of incubation time, temperature, and seminal plasma concentration was investigated. Physical properties of the active seminal plasma substance were characterized by studying effects of dialysis and ultracentrifugation. The origin of the active seminal plasma substance was investigated by studying activity of autopsy materials from male accessory glands. Identity of cells that are influenced by seminal plasma activity was investigated by using two-color flow cytometry with monoclonal antibodies specific for Fc gamma RIII and different phenotypic markers of leukocytes. RESULTS: Incubation of lymphocytes in seminal plasma significantly increased percentages of cells expressing Fc gamma RIII. Maximal increases were observed after seminal plasma incubation at 37 degrees C for 90 to 120 minutes and increases were significantly correlated with seminal plasma concentration. Seminal plasma activity was not altered by ultracentrifugation (100,000 x g for 30 minutes) but completely removed by dialysis (12,000 to 14,000 pore size). Fc gamma RIII-positive lymphocytes markedly increased after incubation in prostatic but not in seminal vesicle secretions. Two-color flow cytometry showed that increases of Fc gamma RIII-expressing cells occurred within the subset of CD56-positive natural killer (NK) cells. CONCLUSIONS: Dialyzable compounds of prostatic origin induce significant increases of NK cells expressing Fc gamma RIII. These findings might reflect a novel regulatory mechanism acting on CD56-positive cells within the female reproductive tract after insemination.

Analysis of Variance↗

PubMed2006

Sedimentation velocity analysis of amyloid oligomers and fibrils.

The different aggregation states of amyloid oligomers and fibrils have been associated with distinct biological properties and disease pathologies. These various amyloid species are distinguished by their different molecular weights and sedimentation coefficients and can be consistently resolved, separated, and analyzed using sedimentation velocity techniques. We first describe the theoretical background and use of the preparative ultracentrifuge to separate amyloid fibrils and their oligomeric intermediates from monomeric subunits as well as the factors and limits involved in such methods. The approach can be used to monitor the kinetics of fibril formation as well as providing purified fractions for functional analysis. Secondly, we describe the use of analytical ultracentrifugation as a precise and robust system for monitoring the rate of sedimentation of amyloid fibrils under different solution conditions. Sedimentation velocity procedures to characterize the size, interactions, and tangling of amyloid fibrils as well as the binding of nonfibrillar components to form heterologous complexes are detailed.

Amyloid↗

PubMed2002

Purification of turkey coronavirus by Sephacryl size-exclusion chromatography.

Sephacryl S-1000 size-exclusion chromatography was used to purify turkey coronavirus (TCoV) from infected turkey embryo. TCoV was propagated in the 22-day-old turkey embryos. Intestines and intestinal contents of infected embryos were harvested and homogenized. After low speed centrifugation, the supernatant was concentrated by ultracentrifugation through a cushion of 30 or 60% sucrose solution, or by ammonium sulfate precipitation. The purification methods included sucrose gradient and Sephacryl S-1000 size-exclusion chromatography. Ultracentrifugation through a cushion of 60% sucrose solution was better than the other two methods for concentration of TCoV from intestinal homogenate. The most effective method for purifying TCoV and removing extraneous materials was size-exclusion chromatography as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More spike-rich particles were observed in the sample purified by chromatography than those purified by sucrose gradient as examined by electron microscopy. Differentiation of turkey anti-TCoV antiserum from normal turkey serum was better achieved by ELISA plates coated with TCoV preparation purified by size-exclusion chromatography than that purified by sucrose density gradient. The results indicated that Sephacryl S-1000 chromatography was useful for purification of TCoV.

Animals↗

PubMed1987

Active immunization of NMRI mice against Serratia marcescens. II. K-antigen extracts.

The minimal active immunogenic doses (intraperitoneal administration) of crude K-antigen extracts of Serratia marcescens for NMRI mice were 80 ng. Following ultracentrifugation (300,000 x g, 16 h), supernatant fluids of three K-antigen extracts were free of contaminating DNA, RNA, and 2-keto-3-deoxy-D-manno-octonic acid (KDO), and the contents of Limulus amoebocyte lysate-reactive lipopolysaccharide (LPS) had been reduced from 100- to 1000-fold. The minimal active immunogenic doses of two ultracentrifuged K-antigen extracts were 2 and 10 micrograms, respectively. A mucoid strain of S. marcescens (SM 20-M; serotype O6/O14:H12) yielded nonmucoid (= SM 29-NM) variants that had lost most of the O6/O14 O-antigen (LPS) and all of the SM 29-M K-antigen extract reactivity (ELISA test) and which were ca. 5-fold less mouse-virulent. Crude K-antigen extracts from S. marcescens strain SM 29-M and variant SM 29-NM failed to protect NMRI mice against strain SM 29-M.

Animals↗

PubMed1995

Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions.

Topoisomerase II overexpressed in yeast was purified to near homogeneity. The milligram amounts of active enzyme obtained allowed its study by joint UV-circular dichroism, ultracentrifugation, and biological assays at different protein and salt conditions. First, sedimentation equilibrium was preferred over the other analytical ultracentifuge methods as it is based on firm theoretical grounds and does not require assumptions about the shape of the molecule. The tendency of topoisomerase II to self-associate into dimers was confirmed and shown to depend on both the enzyme concentration and the concentration of salt used. Analysis at five initial protein concentrations (from 0.08 to 1.05 mg/mL, i.e., 0.5-65 microM) provided evidence for a single monomer-dimer equilibrium characterized at 150 mM KCl and 20 degrees C by an association constant, Ka, of approximately 4.8 10(5) M-1 and a delta G degree of approximately -7.5 kcal mol-1. Under these conditions, for a topoisomerase II concentration of 0.08 mg/mL (i.e., 0.5 microM) in the ultracentrifuge cell, almost 80% of the enzyme were found dissociated. Increase of KCl (from 80 to 400 mM) in the medium provoked a continuous change of the association equilibrium so that a value of Ka approximately 10(5) M-1 corresponding to delta G degree approximately -7 kcal mol-1 was found for topoisomerase II in 400 mM KCl at 20 degrees C. Second, circular dichroism (CD) showed the sensitivity of the topoisomerase II secondary structure to salt concentration, the observed variations being apparently dependent upon the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis↗

PubMed1995

Expression and physicochemical characterization of human proliferating cell nuclear antigen.

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-200, and hydroxylapatite chromatography. Approximately 20 mg of PCNA was isolated from E. coli cells derived from 2 L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase delta activity at least 25-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 102,000 and a Stokes radius of 37 Angstrum by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96,800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a dimeric species at low dilution. Cross-linking experiments revealed the presence of PCNA dimers which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a dimer at low protein concentrations.

Base Sequence↗

PubMed1992

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Spectroscopic and thermodynamic characterization of the transcription antitermination factor NusE and its interaction with NusB from Mycobacterium tuberculosis.

N-utilizing proteins (Nus) form a complex involved in the regulation of rRNA biosynthesis in enteric bacteria by modulating the efficiency of transcriptional termination [Nodwell, J. R., and Greenblatt, J. (1993) Cell 72, 261-268]. The protein NusE (identical to the protein S10 of the small ribosomal subunit) from the pathogenic mycobacterium M. tuberculosis has been cloned and overexpressed in Escherichia coli. The pure protein has been characterized by circular dichroism, ultracentrifugation, NMR, and binding to NusB. The near-ultraviolet circular dichroism spectrum of this protein suggests that it has a moderate (ca. 12-16%) alpha-helical content at 30 degrees C. The protein undergoes cold denaturation, with a temperature of maximum stability near 40 degrees C, implying a substantial heat capacity difference between the folded and unfolded states. The sedimentation equilibrium and velocity data indicate that the protein is monomeric and expanded in solution. NMR spectroscopy shows that there is no significant tertiary structure, and confirms the low secondary structure content at low temperatures. Furthermore, there was evidence for more structure at 30 degrees C than at 10 degrees C. Well-defined shifts in peaks in the HSQC spectrum of (15)N labeled NusE/NusB when the unlabeled counterpart was added at approximately stoichiometric concentrations showed the formation of a NusE-NusB complex in the absence of RNA. The far-UV CD and ultracentrifuge experiments, however, indicated relatively weak binding. Isothermal titration calorimetry showed the binding was weak and endothermic at 15 degrees C, with a total DeltaH of > or =10 kcal/mol. This weak binding is consistent with a small interaction interface and lack of large conformational rearrangements in the predominantly unfolded NusE protein. The conformational flexibility of NusE may be important for its roles in both the ribosome and antitermination complexes.

Amino Acid Sequence↗

PubMed2001

Distal ligand reactivity and quaternary structure studies of proximally detached hemoglobins.

The linkage between the proximal histidines and the proximal polypeptide in normal adult human hemoglobin (Hb A) has been proposed to play a major role in transmitting allosteric effects between oxygen binding sites [Perutz, M. F. (1970) Nature 228, 726-734]. Here we present circular dichroism (CD), (1)H NMR, analytical ultracentrifugation, and stopped-flow kinetic data to better define the quaternary structure of hemoglobins in which the linkage between the proximal histidines and the polypeptide backbone has been broken [Barrick et al. Nat. Struct. Biol. 4, 78-83 (1997)] and to characterize the distal ligand binding properties of these proximally detached Hbs. CD spectroscopy indicates that rHb (alphaH87G) and rHb (alphaH87G/betaH92G) retain at least partial T-quaternary structure with distal ligand bound, whereas rHb (betaH92G) does not, consistent with (1)H NMR spectra. Analytical ultracentrifugation reveals significant tetramer dissociation in rHb (betaH92G) to be the likely cause of loss of T-state markers. These quaternary structure studies indicate that in distally liganded Hb, the T-state is compatible with proximal linkages in the beta- but not the alpha-chains. (1)H NMR titrations of rHb (alphaH87G) with n-butyl isocyanide demonstrate the alpha-chains to be of high affinity as compared with the beta-chains. Comparing ligand association and dissociation rates between the rHb (alphaH87G) variant with the T- and R-states of wild-type Hb A indicates that at the alpha-chains, carbon monoxide affinity is modulated entirely by the proximal linkage, rather than from distal interactions. Some residual allosteric interactions may remain operative at the beta-chains of rHb (alphaH87G).

Allosteric Regulation↗

PubMed1991

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.

Animals↗

PubMed1988

Characterization of dodecylphosphocholine/myelin basic protein complexes.

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.

Animals↗

PubMed1975

The quaternary structure of citrate synthase from Escherichia coli K12.

A combination of equilibrium ultracentrifugation and polyacrylamide gel electrophoresis techniques has been used to establish the quaternary structure of citrate synthase from acetate-grown Escherichia coli K12 3000. In polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS), the pure enzyme showed one major band whose mobility was consistent with a molecular weight of 46,000 plus or minus 2000 g/mol, and a little material of 87,000 plus or minus 5000 g/mol. When first cross-linked with dimethyl suberimidate and then submitted to electrophoresis in SDS, citrate synthase showed six bands, in widely different amounts, whose apparent molecular weights were almost integral multiples of 47,000 g/mol. The dimer was the major product of the cross-linking procedure. In 6 M guanidine HCl at pH 7.0, citrate synthase behaved as a single component in high-speed sedimentation equilibrium experiments, with a weight average molecular weight of 43,400 plus or minus 300 g/mol. The molecular weight of native citrate synthase was investigated by high-speed sedimentation equilibrium ultracentrifugation under different conditions of pH and KCl concentration. In 0.02 M Tris-Cl at pH 7.0 and 7.8, the enzyme was a mixture of oligomers, with species ranging from monomer (47,000 g/mol) to greater than decamer being present. At pH 9.0, only dimer was seen (94,000 g/mol). Large aggregates were present at pH 10.0. The addition of small amounts of KCl, a potent activator of the enzyme, simplified the mixture of oligomers considerably at pH 7.8. A detailed analysis of the data with 0.05 M KCl indicated that dimer and hexamer were the only species present, with marked nonideality. Increasing the KCl concentration to 0.10 M converted all the enzyme to hexamer. The amino acid composition of E. coli citrate synthase was presented. Taken together with peptide mapping experiments of others (J. A. Wright and B. D. Sanwal (1971), J. Biol. Chem. 246 1689), it indicates that the subunits have all the same or very similar amino acid sequences. The dansylation method revealed only methionine at the N-termini of the citrate synthase polypeptide chains. Citrate synthase from E. coli thus resembles the enzyme from eukaryotes in that it consists of subunits weighing just under 50,000 g/mol, although these subunits are more highly aggregated in the bacterial enzyme under most conditions. This conclusion is in disagreement with that of Wright and Sanwal (1971, see above), who reported a subunit size of 62,000 g/mol.

Amino Acids↗

PubMed2002

Hydrodynamic properties and quaternary structure of the 90 kDa heat-shock protein: effects of divalent cations.

The 90 kDa heat-shock protein (Hsp90) is one of the major stress proteins whose overall structure remains unknown. In this study, we investigated the influence of divalent cations Mg(2+) and Ca(2+) on the hydrodynamic properties and quaternary structure of Hsp90. Using analytical ultracentrifugation, size-exclusion chromatography, and polyacrylamide gel electrophoresis, we showed that native Hsp90 was mostly dimeric. The Hsp90 dimer had a sedimentation coefficient, s(w,20) degrees, of 6.10 +/- 0.03 S, which slightly deviated from the hydrodynamics of a globular protein. Using chemical cross-linking and analytical ultracentrifugation, we showed that Mg(2+) and Ca(2+) induced a tertiary conformational change of Hsp90, leading to a self-association process. In the presence of divalent cations, Hsp90 existed as a mixture of monomers, dimers, and tetramers at equilibrium. Finally, to identify Hsp90 domains involved in this divalent cation-dependent self-association, we studied the oligomerization state of the N-terminal (positions 1-221) of Hsp90, the influence of an N-terminal specific ligand, geldanamycin (GA), and the effect of C-terminal truncation on the ability of Hsp90 to oligomerize in the presence of divalent cations. We previously showed that GA inhibits Hsp90 heat-induced oligomerization [Garnier, C., Protasevich, I., Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys. Res. Commun. 249, 197-201], but now we observed that GA does not influence divalent cation-dependent oligomerization of Hsp90, suggesting another mechanism. This mechanism involved the C-terminal part of the protein since C-terminally truncated Hsp90 did not oligomerize in the presence of divalent cations.

Animals↗