The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.     

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.     

Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Some suggested polymer subunit compositions.

1. Some of the individual members of the polymeric series of proteins from human haptoglobin types 2-1 and 2-2 were isolated by gel electrophoresis. By reacting this purified material with less than an equivalent amount of hemoglobin and analyzing the result by electrophoresis, the number of haptoglobin-hemoglobin complexes could be clearly counted. For the haptoglobin 2-1 series, the number of complexes formed was n+1, where n is the serial order, in decreasing electrophoretic mobility, of the haptoglobin polymeric form used. For the haptoglobin 2-2 series, the number of complexes was n+2. 2. For the first three members of haptoglobin 2-1 series, the haptoglobin-hemoglobin composition of the complexes was estimated from scans of the unstained gels. The data indicated that this series consists of 2,3,4... alpha beta haptoglobin subunits, each of which can combine with an alpha beta subunit of hemoglobin.     

Ligand binding to symmetrical FeZn hybrids of variants of human HbA with modifications in the alpha1-beta2 interface

The equilibria of oxygen binding to and kinetics of CO combination with the symmetrical iron-zinc hybrids of a series of variants of human adult hemoglobin A have been measured at pH 7 in the presence of inositol hexaphosphate (IHP). In addition, the kinetics of CO combination have also been measured in the absence of IHP. The hybrids have the heme groups of either the alpha or the beta subunits replaced by zinc protoporphyrin IX, which is unable to bind a ligand and is a good model for permanently deoxygenated heme. The variants examined involve residues located in the alpha1beta2 interface of the hemoglobin tetramer. Alterations of residues located in the hinge region of the interface are found to affect the properties of both the alpha and the beta subunits of the protein. In contrast, alterations of residues in the switch region of the interface have substantial effects only on the mutant subunit and are poorly communicated to the normal partner subunit. When the logarithms of the rate constants for the combination of the first CO molecule with a single subunit in the presence of IHP are analyzed as functions of the logarithms of the dissociation equilibrium constants for the binding of the first oxygen under the same conditions, a linear relationship is found. The relationship is somewhat different for the alpha and beta subunits, consistent with the well-known differences in the geometries of their ligand binding sites.     

Light-scattering investigation of the subunit structure and dissociation of Helix pomatia hemocyanin. Effects of salts and ureas

The effects of various salts of the Hofmeister and aliphatic acid salt series and hydrophobic reagents of the urea series on the subunit structure and the dissociation of Helix pomatia alpha-hemocyanin were investigated by employing light-scattering molecular weight methods. In moderate ranges of salt concentrations [0-1.0 M NaClO4, NaSCN, NaI, and guanidinium chloride (GdmCl) and 0-2.0 M NaBr], the dissociation reaction is essentially a two-step process characterized by the dissociation of whole hemocyanin molecules dissociating to half-molecules of decamers followed by the dissociation of the half-molecules to five dimeric fragments. The effectiveness of the salts and relative ineffectiveness of the ureas and GdmCl as dissociating agents in the first step of the dissociation reaction suggest that the stabilization of the contact areas between half-molecules in solution is largely a nonhydrophobic energy process involving polar and ionic interactions. Hydrophobic forces appear to be important, however, for stabilization of the half-molecules through side to side contacts of the five dimeric units that make up each half-molecule. The analysis of our dissociation data by use of equations derived in our previous studies [Herskovits, T. T., & Harrington, J.P. (1975) Biochemistry 14, 4964-4971] gave apparent estimates of amino acid groups of about 60-150 for each of the contact areas between the cylindrically shaped half-molecules and 30-60 for each of the dimers in the half-molecules themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-scattering investigation of the dissociation behavior of Lunatia heros and Littorina littorea hemocyanins

The subunit structure and dissociation of the hemocyanins of two marine snails, Lunatia heros and Littorina littorea, were investigated by light-scattering molecular weight methods. The hemocyanins of both species of snails are readily dissociated to fragments of one-tenth and one-twentieth of the parent proteins of close to 9 X 10(6) daltons by either increasing the pH or using dissociating reagents of the hydrophobic urea series or some of the Hofmeister salts. The lower members of the latter group of reagents, NaCl, and to some extent also NaBr were found to have only marginal effects on the observed molecular weight transitions, suggesting that the two hemocyanins investigated possess beta-type subunits, which are known to be resistant to NaCl dissociation. The molecular weight profiles obtained with the various dissociating reagents were single inverted sigmoidal-shaped curves for both Lunatia and Littorina hemocyanins, suggesting overlapping transitions. The ultracentrifugation patterns and the species-distribution plots based on the urea dissociation data of Littorina hemocyanin suggest the presence of whole, half, and one-tenth molecular weight species in the dissociation transition region. Fitting of the urea dissociation data of Littorina hemocyanin obtained at both pH 5.7 and pH 8.0, assuming a sequential two-step dissociation scheme used in our previous studies [Herskovits, T. T., & Russell, M. W. (1984) Biochemistry 23, 2812-2819], was found to be consistent with a model of a few hydrophobic binding sites at the contact areas of the half-molecules and a much larger apparent number of binding sites (Napp) at the side to side contacts of the one-tenth molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit dissociations in natural and recombinant hemoglobins.

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.     

Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA

Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.     

The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

Dissociation of the sodium-ion-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and reconstitution of the active complex from the isolated subunits

Oxaloacetate decarboxylase was reconstituted from the purified alpha subunit and a Triton X-100 extract of bacterial membranes devoid of this protein. Upon freezing of oxaloacetate decarboxylase in salt solutions, the enzyme was split into subunits and the catalytic activity was abolished. The catalytically active decarboxylase complex was reconstituted by decreasing the salt concentration of the dissociated sample. The conditions for the inactivation were critical for an optimum recovery of catalytically active enzyme during reconstitution, and modest dissociating conditions generally improved the yield of the reconstitutively active decarboxylase. The dissociated enzyme has been separated by chromatography on avidin-Sepharose into two fractions: fraction I, that was not retained on the column, consisted of the beta + gamma subunits, and fraction II consisted of the biotin-containing alpha subunit. Oxaloacetate decarboxylase was reconstituted from a mixture of the isolated alpha and beta + gamma subunits. The Na+ transport activity was recovered, if a mixture of subunits alpha and beta + gamma was incorporated into liposomes, or by a sequential reconstitution, starting with the formation of proteoliposomes with the integral membrane proteins beta + gamma and completed by an attachment of the peripheral subunit alpha.     

G alpha i-G alpha s chimeras define the function of alpha chain domains in control of G protein activation and beta gamma subunit complex interactions

Gs and Gi2 are G proteins whose alpha subunits are 65% homologous. Within the 355 amino acid alpha i2 polypeptide, substitution of residues Ile213-Lys319 with the corresponding alpha s region (Ile235-Arg356) generated a chimera that activated adenylyl cyclase, indicating that the alpha s activation domain resides within this 122 amino acid alpha s sequence. Mutation within alpha s residues Glu15-Pro144 resulted in an alpha s polypeptide having an enhanced rate of GDP dissociation. Mutation within two regions of the N-terminus influenced the ability of pertussis toxin to ADP-ribosylate the alpha subunit polypeptide, a reaction controlled by the beta gamma subunit complex. The findings define the G protein alpha subunit N-terminus as a regulatory region controlling beta gamma subunit interactions and GDP dissociation independent of the GTPase and effector activation domains.     

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.     

Multiple replacements at position 211 in the alpha subunit of tryptophan synthase as a probe of the folding unit association reaction

Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the alpha subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding. Previous studies [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965-2974] have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units. In terms of the secondary structure, the amino folding unit consists of the first six strands and five alpha helices of this alpha/beta barrel protein. The carboxy folding unit comprises the remaining two strands and three alpha helices; position 211 is in strand 7. Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly. In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation. The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles. When considered with previous data on replacements at other positions in the alpha subunit [Hurle, M. R., Tweedy, N. B., & Matthews, C. R. (1986) Biochemistry 25, 6356-6360], it is clear that beta strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process.     

Kinetic studies on the role of elongation factors 1 beta and 1 gamma in protein synthesis

An equilibrium isotope exchange technique was used to measure in an Artemia system the catalytic influence of elongation factor (EF) 1 beta gamma on the dissociation of GDP from the complex of elongation factor 1 alpha.[3H] GDP in the presence of an excess of free GDP. The kinetic data demonstrate that, in analogy to procaryotes, dissociation of GDP occurs via the formation of a transient ternary complex of EF-1 alpha.GDP.EF-1 beta gamma. The rate constants for the dissociation of GDP from EF-1 alpha.GDP and from the ternary complex EF-1 alpha.GDP.EF-1 beta gamma were found to be 0.7 x 10(-3) and greater than or equal to 0.7 s-1, respectively. The equilibrium association constants of GDP to EF-1 alpha.EF-1 beta gamma and of EF-1 beta gamma to EF-1 alpha.GDP were found to be 2.3 x 10(5) and 4.2 x 10(5) M-1, respectively. Judged from the known elongation rate in vivo and kinetic constants of nucleotide exchange, it was estimated that the recycling of EF-1 alpha may be a rate-controlling step in eucaryotic translation. As a model for GTP exchange, the formation of the ternary EF-1 alpha.guanylyl (beta gamma-methylene)diphosphonate.EF-1 beta gamma complex was also studied. It was observed that both an increase of the level of aminoacyl-tRNA and of temperature favored the dissociation of this complex, thereby enabling EF-1 beta gamma to recycle as a catalyst. This behavior would explain the frequent occurrence of a heavy form of elongation factor 1 in extracts of the eucaryotic cell.     

Involvement of Glu G3(101)beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins

The glutamyl residue at G3(101)beta of normal hemoglobin (Hb A) is one of the alpha 1 beta 2 subunit contacts which are vital to O2 binding properties of the molecule. The O2 equilibrium properties of the four mutants with different substitutions at this site are studied in order to elucidate the role of this residue. Under stripped conditions with minimum chloride the order of O2 affinity is: Hb A (Glu) much less than Hb Rush (Gln) less than or equal to Hb British Columbia (Lys) less than or equal to Hb Potomac (Asp) less than or equal to Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K4, are similar, indicating that the allosteric constants (L) of these mutants are greatly reduced. Therefore, the G3(101)beta residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M Cl- and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their O2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. Hb Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr effect and a smaller heat of oxygenation at pH 6.5. These changes are consistent with an increased positive net charge in the central cavity of Hb Rush and subsequent extra anion binding in the deoxy form. The tetramer to dimer dissociation constants are estimated to be greater than normal for Hb British Columbia and less than normal for Hb Alberta. This comparative study of the G3(101)beta mutants indicates that the size and the charge of this residue may influence the switching of two neighboring interchain hydrogen bonds that occurs during oxygenation of normal hemoglobin.     

Effect of phosphate on the kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains

The kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains was investigated under various buffer conditions by use of a circular dichroism (CD) stopped-flow apparatus. The difference CD spectra of hemoglobin against its constituent chains were independent of the buffer conditions, while the time courses of the Soret CD after mixing equimolar amounts of the alpha and beta chains changed with the buffer conditions. The time courses were analyzed on the basis of a scheme which included a monomer-tetramer equilibrium of the beta chain (beta 4 in equilibrium 4 beta), dissociation of the beta 4 (beta 4 leads to 4 beta), and a second-order combination of alpha and beta monomers (alpha + beta leads to alpha beta). The analysis showed that buffer conditions affected the dissociation of the beta 4 rather than the monomer combination: The rate of the dissociation of the beta 4 accelerated with decreasing phosphate concentration, while the rate of the monomer combination was less sensitive to the phosphate concentration. This result indicates that the stability of the beta 4 depends on the phosphate concentration. It was furthermore suggested that the inorganic phosphate was bound to the beta 4 with an association constant of 133 M-1 and a Hill coefficient of 1.2.     

Structure/function analysis of the integrin beta 1 subunit by epitope mapping

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti- chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.