Structure of alpha 2-macroglobulin from the arthropod Limulus polyphemus.

A structural and functional homologue of vertebrate alpha 2-macroglobulin (alpha 2M) has been identified in the hemolymph and blood cells of the arthropod Limulus polyphemus, one of the oldest living fossil invertebrates (Quigley, J. P., and Armstrong, P. B. (1985) J. Biol. Chem. 260, 12715-12719). The subunit molecular mass is 185 kDa. The native molecular mass, determined by scanning transmission electron microscopy (STEM) under conditions in which the linear relationship between the STEM large angle detector signal and specimen mass thickness allows the determination of the total macromolecular mass, was 354 +/- 35 kDa. Sedimentation equilibrium measurements gave a value of 366 kDa, independent of solute concentration. Sedimentation velocity experiments indicated a homogeneous component with a frictional ratio of 1.41. Thus, the native structure appears to be a dimer, with a somewhat extended conformation. The behavior during gel permeation chromatography was anomalous, yielding an apparent molecular mass approximately half-way between that expected for the dimeric and tetrameric configurations. Transmission electron microscopy of negatively stained preparations revealed a dimeric butterfly-like structure that collapsed following reaction with chymotrypsin.     

Molecular weights of apoprotein B obtained from human low-density lipoprotein (apoprotein B-PI) and from rat very low density lipoprotein (apoprotein B-PIII)

Human low-density lipoproteins (LDL) were isolated from single donors by differential centrifugation between densities of 1.020 and 1.050 g/mL. The LDL were reduced and alkylated in 7 M guanidine hydrochloride, and the lipid was removed by multiple extractions in the cold with a mixture of diethyl ether and ethanol. Sedimentation studies on the resultant human apoprotein B (apoprotein B-PI) at low concentrations in 6.00 M guanidine hydrochloride showed a single sharp boundary with a sedimentation coefficient of 2.15 +/- 0.04 S at 25 degrees C, uncorrected for viscosity or density. Diffusion experiments performed in the same solvent at low speeds in the analytical ultracentrifuge gave a D25 = 0.694 +/- 0.043 Fick. Combining these values with an apparent specific volume of 0.703 mL/g yielded a molecular weight of 387 000, indistinguishable from that obtained by sedimentation equilibrium analysis in 7 M guanidine hydrochloride. Similar values were also obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rat very low density lipoproteins (VLDL), isolated from sera of Triton WR1339 treated animals, were used as the source of rat apoprotein B-PIII. The delipidated VLDL were solubilized in sodium dodecyl sulfate, and apoprotein B-PIII was isolated by Sepharose 4B chromatography. With appropriate corrections for density and viscosity, the behavior of rat apoprotein B-PIII was identical, upon analytical ultracentrifugation, in 6 and 7.7 M guanidine hydrochloride, corresponding to sedimentation and diffusion coefficients of 1.47 S and 0.92 Fick, respectively, in 6 M guanidine hydrochloride. These data may be combined to yield a molecular weight of 210 000. Similar values were obtained by calibrated sedimentation analysis, by Sepharose 2B chromatography in guanidine hydrochloride, and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.     

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.     

Effect of temperature on the self-assembly of the Escherichia coli ClpA molecular chaperone

Protein quality control pathways rely upon ATP-dependent proteases, such as Escherichia coli ClpAP, to perform maintenance roles in the cytoplasm of the cell. ATP-dependent proteases remove misfolded and partially synthesized proteins. This action is particularly important in situations where an unregulated accumulation of such proteins will have a deleterious effect on the cell. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and the ATPase/protein unfoldase ClpA (84.2 kDa monomer). ClpA also uses its protein unfolding activity to remodel proteins and protein complexes; thus, in the absence of the proteolytic component, ClpA is considered a molecular chaperone. Previous reports, by others, suggested that ClpA exists in a monomer-dimer equilibrium at 4 °C. In contrast, using a combination of sedimentation velocity, sedimentation equilibrium, and dynamic light scattering, we recently reported that ClpA exists in a monomer-tetramer equilibrium at 25 °C. Here we report an investigation of the effect of temperature on the self-association of the E. coli ClpA protein unfoldase using analytical ultracentrifugation techniques. The results of sedimentation velocity and sedimentation equilibrium experiments performed at multiple loading concentrations of ClpA over a range of temperatures from 3.9 to 38.2 °C are discussed. Sedimentation velocity experiments show a decrease in weight average s(20,w) at the extremes of temperature. This result, along with extensive sedimentation equilibrium data and analysis, suggests the presence of a dimeric intermediate of ClpA that is differentially populated as a function of temperature. Further, analysis of sedimentation equilibrium data as a function of temperature led us to propose a monomer-dimer-tetramer equilibrium to describe the temperature dependence of ClpA self-assembly in the absence of nucleotide.     

Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.

The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E. coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed.     

Quaternary structure of delta-aminolevulinic acid synthase from Rhodopseudomonas spheroides.

Delta-Aminolevulinic acid synthase (succinyl-CoA: glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37) was purified from Rhodopseudomonas spheroides. The purity of the enzyme preparation was established by its behavior in disc electrophoresis in the presence and absence of sodium dodecyl sulfate and by analytical ultracentrifugation. The molecular weight of the enzyme as determined by sedimentation equilibrium was found to be about 80,300, a value similar to those obtained by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient centrifugation. The molecular weight of the enzyme, denatured with either sodium dodecyl sulfate or guanidine hydrochloride, was found to be about 45,000 and 41,000, respectively. The dimeric structure was supported by sedimentation in sucrose gradients. Further evidence for the dimetic nature of the enzyme was obtained by gel electrophoresis of the enzyme treated with dimethylsuberimidate and sodium dodecyl sulfate.     

Analytical ultracentrifugation studies of translin: analysis of protein-DNA interactions using a single-stranded fluorogenic oligonucleotide.

Translin is a recently identified nucleic acid binding protein that appears to be involved in the recognition of conserved sequences found at many chromosomal breakpoints. Previous reports indicate that, based on gel filtration analysis and electron microscopy of protein-DNA complexes, translin forms an octameric structure that binds the DNA. In this study, we further examine the possibility of self-association of translin and its interactions with DNA by analytical ultracentrifugation. Sedimentation velocity analysis of translin indicates that the predominant species sediments with a sedimentation coefficient of 8.5 S and has a frictional ratio, f/f(omicron), of 1.35; these data are consistent with the presence of an octamer with an ellipsoidal configuration; a small amount of a component with significantly higher mass is also present. Equilibrium sedimentation studies of translin at three different protein concentrations also indicate that the predominant species present is an octamer with a minor fraction of aggregated species. Neither monomer nor dimer was detected. Sedimentation equilibrium studies of translin with an FITC-labeled single-stranded oligonucleotide were performed to examine the interaction. A novel analysis method has been developed to analyze protein-nucleic acid interactions based on global fitting of scans of 280 and 490 nm to appropriate mathematical models. Utilizing this method, it was determined that the DNA binding species of translin is an octamer binding a single-stranded oligonucleotide with a DeltaG degrees value of -9.49 +/- 0.12 kcal/mol, corresponding to a dissociation constant, K(d), of 84 +/- 17 nM. On the basis of this evidence and electron microscopy, it is envisioned that translin forms an annular structure of eight subunits, hydrodynamically an oblate ellipsoid, which binds DNA at chromosomal breakpoints.     

Staphylococcus aureus sortase A exists as a dimeric protein in vitro

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.     

Behavior of Tn3 resolvase in solution and its interaction with res

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.     

Colloidal properties of human transferrin receptor in detergent free solution

The colloidal properties of transferrin receptor, isolated from human placenta, in detergent free solution has been investigated by light scattering techniques and analytical ultracentrifugation. In detergent free solution at 293.2 K, hTfR forms stable aggregates with an apparent hydrodynamic radius of 17 nm. The molecular mass was determined by ultracentrifugation to lie between (1722+/-87) kDa (sedimentation equilibrium) and (1675+/-46) kDa (sedimentation velocity). This implies that the aggregates are build up from nine hTfR dimers. Based on model calculations, which are in good agreement with the experimental data, we propose a torus-like structure for the aggregates. Upon pH shift from pH 7.5 to 5.0 or removal of the N-linked carbohydrate chains, formation of larger aggregates is induced. These aggregates can be described in terms of porous fractal structures. We propose a simple model, which accounts for that behaviour assuming that the aggregation is mainly due to the reduction of negative surface charge.     

Caldesmon. Molecular weight and subunit composition by analytical ultracentrifugation

A wide range of values has been reported for the subunit and molecular weights of smooth muscle caldesmon. There have also been conflicting reports concerning whether caldesmon is a monomer or dimer. We attempted to resolve these uncertainties by determining the molecular weight of chicken gizzard smooth muscle caldesmon using the technique of sedimentation equilibrium in the analytical ultracentrifuge. Unlike previous methods that have been used to estimate the molecular weight of caldesmon, the molecular weight determined by equilibrium sedimentation does not depend upon assumptions about the shape of the molecule. We concluded that caldesmon in solution is monomeric with a molecular mass of 93 +/- 4 kDa, a value that is much less than those previously reported in the literature. This new value, in conjunction with sedimentation velocity experiments, led to the conclusion that caldesmon is a highly asymmetric molecule with an apparent length of 740 A in solution. The mass of a cyanogen bromide fragment, with an apparent mass of 37 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was determined to be 25.1 +/- 0.6 kDa using sedimentation equilibrium. These results imply that the reported molecular weights of other fragment(s) of caldesmon have also been overestimated. We have determined an optical extinction coefficient for caldesmon (E1%(280 nm) = 3.3) by determining its concentration from its refractive index which was measured in the analytical ultracentrifuge. From the above values of the molecular weight and the extinction coefficient, we redetermined that the caldesmon molecule has two cysteines and recalculated the stoichiometric molar ratio of actin/tropomyosin/caldesmon in the smooth muscle thin filament to be 28:4:1.     

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.     

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.     

Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

Turkey gizzard caldesmon: molecular weight determination and calmodulin binding studies

Sedimentation equilibrium and sedimentation velocity measurements demonstrate that turkey gizzard caldesmon is an elongated molecule of molecular mass 75 +/- 2 kDa. The frictional ratio (2.14) is consistent with a prolate ellipsoid of axial ratio 24, corresponding to an apparent length and width of 516 and 21.5 A, respectively. As was previously determined for chicken gizzard caldesmon [Graceffa, P., Wang, C.-L.A., & Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202], this molecular weight is appreciably smaller than the value (approximately 135,000) estimated from the results of NaDodSO4 gel electrophoresis experiments. However, a significant difference between the true molecular weights of turkey and chicken gizzard caldesmons--75,000 versus 93,000--also points to probable molecular weight variations within the subclass. Binding measurements, based on perturbation of the intrinsic tryptophan fluorescence of caldesmon in the presence of calmodulin, show that the interaction between the two proteins is strongly ionic strength and temperature dependent. Dissociation constants of 0.075 and 0.38 microM were determined in solutions containing 0.1 and 0.2 M KCl, respectively, at 24.3 degrees C. Fluorescence emission spectra and fluorescence anisotropy excitation spectra indicate that the tryptophanyl residues of caldesmon are located in solvent-accessible regions of the molecule, where they exhibit a high degree of mobility even when calmodulin is bound.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.