Chromatographic properties of human leukocyte alpha-interferon A on sorbents with sepharose and silochrome matrices

Chromatography on immobilized monoclonal antibodies NK-2 from a bacterial strain-producer resulted in a pure human leukocyte alpha-interferon A (alpha-INF-A) homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reverse phase high performance liquid chromatography. The chromatographic properties of partially purified alpha-INF-A on synthetic and commercial sorbents containing immobilized dyes, aromatic dipeptides, chelating and hydrophobic ligands as well as on porous glass have been investigated. In most cases, [125I]alpha-INF-A was used as an inner standard. The chromatographic behaviour of native and [125I]-labeled alpha-INF-A was practically the same. alpha-INF-A was most effectively chromatographed on porous glass, L-Trp-L-Trp-Sepharose 4B and Cu2+-chelate sorbents. In the latter case, the feasibility of substitution of the Sepharose matrix for the silochrome one has been demonstrated. It has been proposed that alpha-INF-A has a hydrophobic "pocket" with exposed aromatic amino acid residues which are capable of selective binding to aromatic dipeptides.     

Study of the aggregation of membrane proteins of erythrocyte ghosts using SDS-PAAG electrophoresis

Using the method of electrophoresis in SDS-PAAG the authors showed a diminution of proteins of bands I + II (spectrins) and III (major integral protein) after irradiation of erythrocyte ghosts with doses of 50 to 1000 Gy. We failed to ascertain that radiation-induced lipid peroxidation is involved into membrane protein aggregation. Among the radiolysis products, OH-radicals were shown to contribute markedly to the radiation effect observed.     

Dimerization and oligomerization of the chaperone calreticulin.

The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disulfide-bridged dimers under physiological conditions, indicating that the SH group may not be fully accessible in the native protein. Using PAGE, urea gradient gel electrophoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5-6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 m or SDS concentrations above 0.025%. Moreover, we show that calreticulin also has the ability to self-oligomerize through noncovalent interactions at urea concentrations above 2.6 m at pH below 4.6 or above pH 10, at temperatures above 40 degrees C, or in the presence of high concentrations of organic solvents (25%), conditions that favour partial unfolding or an intramolecular local conformational change that allows oligomerization, resulting in a heterogeneous mixture of oligomers consisting of up to 10 calreticulin monomers. The oligomeric calreticulin was very stable, but oligomerization was partially reversed by addition of 8 m urea or 1% SDS, and heat-induced oligomerization could be inhibited by 8 m urea or 1% SDS when present during heating. Comparison of the binding properties of monomeric and oligomeric calreticulin in solid-phase assays showed increased binding to peptides and denatured proteins when calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity.     

Constitutive HSP70: oligomerization and its dependence on ATP binding.

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.     

The effect of nucleotides on oligomeric state of human lactoferrin

A polyfunctional protein lactoferrin (LF) which is present in human barrier fluids, blood and milk and this protein of acute phase is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. Using the methods of small-angle X-ray scattering and light-scattering it was shown that LF in solution exists in oligomeric state. The level of LF oligomerization depends upon its concentration and time of keeping of no frozen neutral protein solutions. At the concentrations comparable with those in human milk (1-6 mg/ml) the average inertial radius values (Rg) of LF can reach 100-450 angstroms, while Rg for monomer LF form is 26.7 angstroms. LF was shown to complex with different nucleotides and hydrolyze them. The addition of ATP and AMP to LF demonstrating any level of oligomerization leads to increase of oligomerization processes and enhancement of the Rg values up to 600-700 angstroms According to different models of LF monomer association to its oligomeric forms (sphere, plate, cylinder) the oligomeric complexes demonstrate high Rg values which can contain from several tens up to several thousands of LF monomers. A possible role of LF oligomerization for different biological functions of the protein is discussed.     

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.     

Characteristics of A-type intracytoplasmic particles produced by human cells

Radioimmunoprecipitation was used to analyse comparatively proteins in cytoplasmic A-type particles (CAP) and structural proteins of D-type virions in Hep-2 system of cultivated human cells. Proteins of CAP were iodinated and studied by electrophoresis in SDS-PAAG. In the preparations obtained, 60 000 (p60), 45 000, 42 000 and 20 000 dalton proteins wee detected. p60 was the main protein in CAP. It was precipitated by purified CAP antiserum rather than by antisera against structural proteins of D-type virions. It was thus demonstrated that the main structural protein of CAP Hep-2 cells contains no antigenic determinants of structural proteins of D-type oncoviruses.     

Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles.

The possible existence of NK-2 receptor subtypes in peripheral smooth muscle preparations from rabbit and hamster was investigated by studying the effect of neurokinin A, the selective NK-2 receptor agonist [beta Ala8] neurokinin A (4-10), the selective NK-2 tachykinin receptor antagonists, MEN 10,376, L 659,877 and R 396, and the pseudopeptide derivative of neurokinin A (4-10), MDL 28,564. All experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each). Both neurokinin A and [beta Ala8] neurokinin A (4-10) produced concentration-dependent contractions of the rabbit isolated bronchus and hamster isolated stomach and colon, as well as enhancement of the nerve-mediated twitches of rabbit isolated vas deferens (pars prostatica). MEN 10,376, L 659,877 and R 396 antagonized the effect of the NK-2 receptor selective agonist in all four tissues under study, although marked differences in antagonist potency were evident for the three antagonists. Thus MEN 10,376 was distinctly more potent (about 100 times) in rabbit than in hamster preparations while L 659,877 and R 396 were more potent in hamster than rabbit preparations. MDL 28,564 showed a distinct agonist character in rabbit preparations while it was virtually inactive in hamster preparations, where it antagonized the effect of the NK-2 receptor selective agonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligomerization-dependent changes in the thermodynamic properties of the TPR-MET receptor tyrosine kinase

Although oligomerization of receptor tyrosine kinases (RTKs) is necessary for receptor activation and signaling, a quantitative understanding of how oligomerization mediates these critical processes does not exist. We present a comparative thermodynamic analysis of functionally active dimeric and functionally inactive monomeric soluble analogues of the c-MET RTK, which clearly reveal that oligomerization regulates the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. Thermodynamic binding data for oligomeric c-MET were obtained from the dimeric TPR-MET oncoprotein, a functionally active fusion derivative of the c-MET RTK. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil dimerization domain from the nuclear pore complex. Comparative data were obtained from a soluble monomeric kinase compromising the c-MET cytoplasmic domain (cytoMET). Significantly, under equilibrium binding conditions, the oligomeric phosphorylated kinase showed a significantly lower dissociation constant (K(d,dimer) = 11 microM) for a tyrosine-containing peptide derived from the C-terminal tail of the c-MET RTK when compared to the phosphorylated monomeric kinase cytoMET (K(d,monomer) = 140 microM). Surprisingly, equilibrium dissociation constants measured for the kinase and ATP were independent of the oligomerization state of the kinase (approximately 10 microM). Stopped-flow analysis of peptide substrate binding showed that the association rate constants (k(2)) differed 2-fold and dissociation rate constants (k(-2)) differed 10-fold when phosphorylated TPR-MET was compared to phosphorylated cytoMET. ATP binding abrogated the differences in k(2) rates observed between the two oligomeric states of the c-MET cytoplasmic domain. These results clearly imply that oligomerization induces important thermodynamic and conformational changes in the substrate binding regions of the c-MET protein and provide quantitative mechanistic insights into the necessary role of oligomerization in RTK activation.     

A single mismatch in the DNA induces enhanced aggregation of MutS. Hydrodynamic analyses of the protein-DNA complexes

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10-12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18-20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh > 500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPgammaS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh > 500 nm in the presence of MutL) as compared to the normal homoduplex (Rh approximately 100-200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system.     

Stages and Conformations of the Tau Repeat Domain during Aggregation and Its Effect on Neuronal Toxicity

Several neurodegenerative diseases are characterized by the aggregation and posttranslational modifications of Tau protein. Its “repeat domain” (TauRD) is mainly responsible for the aggregation properties, and oligomeric forms are thought to dominate the toxic effects of Tau. Here we investigated the conformational transitions of this domain during oligomerization and aggregation in different states of β-propensity and pseudo-phosphorylation, using several complementary imaging and spectroscopic methods. Although the repeat domain generally aggregates more readily than full-length Tau, its aggregation was greatly slowed down by phosphorylation or pseudo-phosphorylation at the KXGS motifs, concomitant with an extended phase of oligomerization. Analogous effects were observed with pro-aggregant variants of TauRD. Oligomers became most evident in the case of the pro-aggregant mutant TauRDΔK280, as monitored by atomic force microscopy, and the fluorescence lifetime of Alexa-labeled Tau (time-correlated single photon counting (TCSPC)), consistent with its pronounced toxicity in mouse models. In cell models or primary neurons, neither oligomers nor fibrils of TauRD or TauRDΔK280 had a toxic effect, as seen by assays with lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. However, oligomers of pro-aggregant TauRDΔK280 specifically caused a loss of spine density in differentiated neurons, indicating a locally restricted impairment of function.     

Homooligomerization of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 occurs before the acquisition of correct intramolecular disulfide bonds and mature immunoreactivity.

The posttranslational maturation of the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza type 3 virus (PIV3) was investigated in pulse-chase experiments in which folding was monitored by immunoprecipitation with conformation-dependent antibodies and gel electrophoresis under nonreducing conditions and oligomerization was monitored by chemical cross-linking and sedimentation in sucrose gradients. The acquisition of mature immunoreactivity and the formation of correct intramolecular disulfide bonds were concurrent events, with half-times of approximately 10 to 15 min. The finding that newly synthesized HN had little reactivity with postinfection cotton rat serum or with most of the members of a panel of HN-specific monoclonal antibodies indicated that the major epitopes of the PIV3 HN protein are highly conformational in nature. Chemical cross-linking studies indicated that the mature HN protein is present in homoligomers, which are probably tetramers. These findings are consistent with recent observations for the HN protein of Sendai virus (S.D. Thompson, W.G. Laver, K.G. Murti, and A. Portner, J. Virol. 62:4653--4660, 1988; S. Vidal, G. Mottet, D. Kolakofsky, and L. Roux, J. Virol. 63:892--900, 1989). Surprisingly, analysis of pulse-labeled HN protein by sedimentation on sucrose gradients after labeling periods of as little as 2 min indicated that it was present intracellularly only in oligomeric form. The same results were obtained when the labeling period was preceded by a 1.5-h cycloheximide treatment to clear the endoplasmic reticulum of presynthesized HN protein, which indicated that the oligomerization did not involve the incorporation of newly synthesized monomers into partially assembled oligomers. Subsequent chase incubations did not significantly alter the sedimentation profile or stability of the oligomeric forms, suggesting that oligomers detected after short labeling periods were tetramers. Association with cellular proteins did not appear to be responsible for the sedimentation of newly synthesized HN protein as an oligomer. The absence of a detectable monomeric form of intracellular HN protein raised the possibility that oligomerization is cotranslational, and it is possible that the type II membrane orientation of the HN protein might be an important factor in its mode of oligomerization.     

Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.     

αB-crystallin polydispersity is a consequence of unbiased quaternary dynamics

The inherent heterogeneity of many protein assemblies complicates characterization of their structure and dynamics, as most biophysical techniques require homogeneous preparations of isolated components. For this reason, quantitative studies of the molecular chaperone αB-crystallin, which populates a range of interconverting oligomeric states, have been difficult, and the physicochemical basis for its polydispersity has remained unknown. Here, we perform mass spectrometry experiments to study αB-crystallin and extract detailed information as to its oligomeric distribution and exchange of subunits under a range of conditions. This allows a determination of the thermodynamic and kinetic parameters that govern the polydisperse ensemble and enables the construction of a simple energy profile for oligomerization. We find that the quaternary structure and dynamics of the protein can be explained using a simple model with just two oligomer-independent interactions (i.e., interactions that are energetically identical in all oligomers from 10mers to 40mers) between constituent monomers. As such, the distribution of oligomers is governed purely by the dynamics of individual monomers. This provides a new means for understanding the polydispersity of αB-crystallin and a framework for interrogating other heterogeneous protein assemblies.     

Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability

Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).     

Multiplicity of molecular forms and thermostability of Torula thermophila UzPT-1 protease]

Alkaline protease preparations with different ratio of molecular forms are isolated from cultural medium of thermophilic fungi Torula thermophila UzPT-1 by means of protein fractionation with (NJ/)2SO4 and gel filtration through Sephadex G-75. The enzyme preparations differ in their thermostability in water at 60 degrees C. High molecular weight oligomeric enzyme forms dissociate in water (at 2-4 degrees C) forming dimeric and monomeric forms. Disaggregation is accompanied by the change in the thermostability of the enzyme preparations. It is concluded that protease thermostability depends on the ratio of dimeric and monomeric forms of the preparation, and it is associated with the conformational state of the enzyme molecules, and it is associated with the conformational state of the enzyme molecules. Oligomeric forms do not dissociate in 1% sodium dodecylsulphate and in 6 M urea. Ca2+ produces dissociation of high molecular weight enzyme forms and the conformational transition into the thermostable state.     

Heterogeneity of streptococcus group A cell wall protein components

Cell wall surface proteins of group A streptococcus (M 29) were isolated by mild chemical extraction with 1 M hydroxylamine pH 6.0 (37 degrees C). The proteins were purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-Trisacryl M. Using two independent methods (disc electrophoresis in 7.5% PAAG pH 8.9 and high pressure gel filtration), it was shown that after chromatography on Sephadex G-150 the original protein fraction contains up to 8 protein components, while SDS-PAAG electrophoresis performed according to Laemmli revealed up to 25 protein components in the same fraction. During SDS-PAAG electrophoresis six protein fractions performed after ion-exchange chromatography were resolved into 40 protein components whose molecular masses vary from 13 to 80 kDa. Possible reasons for the heterogeneity of surface proteins of group A streptococcus cell wall are discussed.     

Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.     

Oligomerization behavior of the archaeal Sm2-type protein from Archaeoglobus fulgidus

As part of a functional analysis of archaeal Sm-related proteins, we have studied the oligomerization behavior of the Sm-2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA-bound heptamer increases at lower pH.     

Physicochemical characterization of the 68 000-dalton protein of bovine neurofilaments

The 68 000-dalton protein from bovine neurofilaments was purified by a combination of chromatography on DEAE-cellulose and on hydroxylapatite in buffers containing 8 M urea. Although the separation of this protein from the other proteins of the neurofilament appeared to be hampered by a mixed association of the several components, a nearly homogeneous product was obtained for study. Sedimentation equilibrium experiments in buffers containing 8 M urea showed the molecule to be a monomer with a molecular weight of 70 600 +/- 2000. Circular dichroic spectra taken under the same conditions gave no evidence of residual alpha-helix. Molecular sieve chromatography in 8 M urea on controlled-pore glass showed that the molecule eluted at an unexpectedly small volume. The small elution volume did not depend significantly on protein concentration and is unlikely to be the result of intermolecular association. Rather, the monomer probably has a conformation more rigid or extended than a classical random coil. When dialyzed into 0.01 M tris(hydroxymethyl)aminomethane/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.1 mM dithioerythritol, pH 8.5, the protein does not assemble into filaments. Sedimentation velocity reveals that under these conditions it consists mainly of a 4.8S molecular species, containing few large particles; sedimentation equilibrium shows that it is composed of oligomers, the smallest present in significant concentration having a molecular weight approximately that of a trimer. Circular dichroism measurements lead to the interpretation that the molecule has refolded in this buffer into a structure that has approximately 55% alpha-helix. Assembly into filamentous particles resembling neurofilaments occurs when the protein is dialyzed against 0.1 M 2-(N-morpholino)ethane-sulfonic acid/0.1% beta-mercaptoethanol/1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/0.17 M NaCl, pH 6.5. We suggest that the oligomeric species present in 0.01 M tris(hydroxymethyl)aminomethane may frequently be present in solubilized preparations of intermediate filaments and may represent an intermediate in the assembly process.