Interaction of hemoglobin and its component alpha and beta chains with band 3 protein

The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

The native energy landscape for interleukin-1beta. Modulation of the population ensemble through native-state topology

A minimalist Go-model, with no energetic frustration in the native conformation, has been shown to describe accurately the folding pathway of the beta-trefoil protein, interleukin-1beta (IL-1beta). While it appears that these models successfully model transition states and intermediates between the unfolded and native ensembles, it is unclear how accurately they capture smaller, yet biologically relevant, structural changes within the native ensemble after energetic perturbation. Here, we address the following questions. Can a simple Go-model of interleukin-1beta, based on native topology, describe changes in structural properties of the native ensemble as the protein stability is changed? Or is it necessary to include a more explicit representation of atoms, electrostatic, hydrogen bonding, and van der Waals forces to describe these changes? The native ensemble of IL-1beta was characterized using a variety of experimental probes under native (0 M NaCl, guanidine hydrochloride (Gdn-HCl)), moderately destabilized (0 M NaCl, 0.8 M Gdn-HCl), and in moderate salt concentration (0.8 M NaCl, 0 M Gdn-HCl). Heteronuclear (1)H-(15)N nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) NMR spectra confirmed that the beta-trefoil global fold was largely intact under these three conditions. However, 25 of the 153 residues throughout the chain did demonstrate (13)C and (1)H-(15)N chemical shifts when perturbed with 0.8 M NaCl or Gdn-HCl. Despite large differences in protection factors from solvent hydrogen-deuterium exchange for all residues between stable (0 M Gdn-HCl) and destabilized (0.8 M Gdn-HCl) IL-1beta, no difference in steady-state (15)N-(1)H NOE enhancements were measured. Thus, the chemical shifts correlate with a global but limited increase in residue flexibility in the presence of Gdn-HCl. Minimalist simulations highlight the regions of greatest position shift between native and 0.8 M Gdn-HCl, which were determined experimentally. This correlation demonstrates that structural changes within the native ensemble of IL-1beta are, at least partially, governed by the principle of minimal energetic frustration.     

Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin.

Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.     

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.     

Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys).

Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A.     

Roles of the beta 146 histidyl residue in the molecular basis of the Bohr effect of hemoglobin: a proton nuclear magnetic resonance study

Assessment of the roles of the carboxyl-terminal beta 146 histidyl residues in the alkaline Bohr effect in human normal adult hemoglobin by high-resolution proton nuclear magnetic resonance spectroscopy requires assignment of the resonances corresponding to these residues. Previous resonance assignments in low ionic strength buffers for the beta 146 histidyl residue in the carbonmonoxy form of hemoglobin have been controversial [see Ho and Russu (1987) Biochemistry 26, 6299-6305; and references therein]. By a careful spectroscopic study of human normal adult hemoglobin, enzymatically prepared des(His146 beta)-hemoglobin, and the mutant hemoglobins Cowtown (beta 146His----Leu) and York (beta 146His----Pro), we have resolved some of these conflicting results. By a close incremental variation of pH over a wide range in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer, a single resonance has been found to be consistently missing in the proton nuclear magnetic resonance spectra of these hemoglobin variants. The spectra of each of these variants show additional perturbations; therefore, the assignment has been confirmed by an incremental titration of buffer conditions to benchmark conditions, i.e., 0.2 M phosphate, where the assignment of this resonance is unambiguous. The strategy of incremental titration of buffer conditions also allows extension of this resonance assignment to spectra taken in 0.1 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane buffer. Participation of the beta 146 histidyl residues in the Bohr effect has been calculated from the pK values determined for the assigned resonances in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer. Our results indicate that the contribution of the beta 146 histidyl residues is 0.52 H+/hemoglobin tetramer at pH 7.6, markedly less than the 0.8 H+/hemoglobin tetramer estimated by study of the mutant hemoglobin Cowtown (beta 146His----Leu) by Shih and Perutz [(1987) J. Mol. Biol. 195, 419-422]. We have found that at least two histidyl residues in the carbonmonoxy form of this mutant have pK values that are perturbed, and we suggest that these pK differences may in part account for this discrepancy. Furthermore, summation of the positive contribution of the beta 146 histidyl residues and the negative contribution of the beta 2 histidyl residues to the maximum Bohr effect measured in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer suggests that additional sites in the hemoglobin molecule account for proton release upon ligation greater than the contribution of the beta 146 histidyl residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and functional similarity between Yersinia pestis capsular protein Caf1 and human interleukin-1 beta

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.     

Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.     

Three-point cross-linking: potential red cell substitutes from the reaction of trimesoyl tris(methyl phosphate) with hemoglobin.

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta 1Val and beta 82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha alpha beta 1(82) greater than B beta 82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha alpha beta 1B beta 82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between pO2 = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.     

Topological frustration and the folding of interleukin-1 beta

The cytokine, interleukin-1beta (IL-1beta), adopts a beta-trefoil fold. It is known to be much slower folding than similarly sized proteins, despite having a low contact order. Proteins are sufficiently well designed that their folding is not dominated by local energetic traps. Therefore, protein models that encode only the folded structure and are energetically unfrustrated (Gō-type), can capture the essentials of the folding routes. We investigate the folding thermodynamics of IL-1beta using such a model and molecular dynamics (MD) simulations. We develop an enhanced sampling technique (a modified multicanonical method) to overcome the sampling problem caused by the slow folding. We find that IL-1beta has a broad and high free energy barrier. In addition, the protein fold causes intermediate unfolding and refolding of some native contacts within the protein along the folding trajectory. This "backtracking" occurs around the barrier region. Complex folds like the beta-trefoil fold and functional loops like the beta-bulge of IL-1beta can make some of the configuration space unavailable to the protein and cause topological frustration.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro

The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.     

The elastase inhibitors of swine serum: isolation and partial characterization of the beta 1-globulin inhibitor and its interaction with elastase

1. The principal elastase inhibitor of swine serum, a beta 1-globulin, has been isolated from serum by ammonium sulfate fractionation, DEAE-cellulose column chromatography and chromatofocusing. 2. The purified beta 1-globulin was homogeneous by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Multiple zones (isoinhibitors) were produced on anionic polyacrylamide gels. The mol. wt for the native, elastase-inactivated inhibitor and the beta 1-globulin-elastase complex were respectively, 65,467 and 60,000 and 79,667. The amino acid residue weight was 63,331. 4. The electrophoretic mobilities of the native inhibitor, elastase-inactivated inhibitor and the inhibitor-elastase complex were respectively, -3.4, -3.8 and -2.2 x 10(-5) cm2/V per sec, the isoelectric points were respectively, 4.78-5.28 (major pIs = 5.15, 5.35), 4.63-5.35 (major pI = 5.13) and 6.02-6.2 (major pI = 6.12). 5. The first order dissociation rate constant for the beta 1-globulin-elastase complex (two-fold molar excess of elastase at 37 degrees C) was 1.9 x 10(-3) per sec with complete dissociation in 40.4 min. The dissociation constant for the complex was 1.47 x 10(-7) M. One mol of elastase was bound per mol of the inhibitor. 6. The beta 1-globulin-elastase complex reacts with antibody to either protein moiety.     

Interleukin-1 beta folding between pH 5 and 7: experimental evidence for three-state folding behavior and robust transition state positions late in folding

The thermodynamic stability and folding kinetics of the all beta-sheet protein interleukin-1beta were measured between 0 and 4 M GdmCl concentrations and pH 5-7. Native interleukin-1beta undergoes a 3.5 kcal/mol decrease in thermodynamic stability, Delta, as pH is increased from 5 to 7. The native state parameter m(NU), measuring protein destabilization/[GdmCl], remains constant between pH 5 and 7, indicating that the solvent-exposed surface area difference between the native state and unfolded ensemble is unchanged across this pH range. Similarly, pH changes between 5 and 7 decrease only the thermodynamic stability, DeltaG(H)2(O), and not the m-values, of the kinetic intermediate and transition states. This finding is shown to be consistent with transition state configurations which continue to be the high-energy configurations of the transition state in the face of changing stability conditions. A three-state folding mechanism U right arrow over left arrow I right arrow over left arrow N is shown to be sufficient in characterizing IL-1beta folding under all conditions studied. The m-values of refolding transitions are much larger than the m-values of unfolding transitions, indicating that that the fast, T(2) (U right arrow over left arrow I), and slow, T(1) (I right arrow over left arrow N), transition states are highly similar to the intermediate I and native state N, respectively. Many of the folding properties of interleukin-1beta are shared among other members of the beta-trefoil protein family, although clear differences can exist.     

Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.

Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins. Both modified proteins carried one molecule of acrylodan each, one at Cys-8 and the other at Lys-103. Neither Cys-71 nor any of the other 13 lysine residues of rIL-1 beta was modified. Cysteine 71 is inaccessible to acrylodan, but the unusual specificity for Lys-103 could be caused by the location of that residue at the bottom of a hydrophobic pocket which might specifically bind the reagent. No double-labeled protein was detected, indicating that the introduction of the label at either site interferes with the labeling at the other. Both acrylodan-modified proteins exhibited bioactivity in the thymocyte proliferation assay at a level equivalent to that of the unmodified control protein (1.7 x 10(7) units/mg), which shows that the modification of either the Cys-8 or Lys-103 position with acrylodan does not interfere with the cellular bioactivities of the respective proteins. Furthermore, receptor binding assays yielded a Kd = 32.0 +/- 4.8 pM for the Lys-103-labeled protein, Kd = 69.5 +/- 12.7 pM for the unmodified protein, and Kd = 75.0 +/- 11.6 pM for the Cys-8-labeled protein. Thus, Cys-8 or Lys-103 modification of rIL-1 beta by acrylodan also does not interfere with the ability of the molecule to bind to its receptor. The slightly higher affinity of the Lys-103-labeled protein for the receptor suggests that the positive charge on this residue in the native molecule may interfere with IL-1 receptor binding. The two fluorescent labeled IL-1 proteins described herein should provide interesting probes for the study of IL-1/IL-1 receptor interactions.     

Treatment with an Interleukin 1 beta antibody improves glycemic control in diet-induced obesity

The proinflammatory cytokine Interleukin 1 beta (IL-1beta) is elevated in obese individuals and rodents and it is implicated in impaired insulin secretion, decreased cell proliferation and apoptosis of pancreatic beta cells. In this study we describe the therapeutic effects by an IL-1beta antibody to improve glucose control in hyperglycemic mice with diet-induced obesity. After 13 weeks of treatment the IL-1beta antibody treated group showed reduced glycated hemoglobin (( *)P=0.049), reduced serum levels of proinsulin (( *)P=0.015), reduced levels of insulin and smaller islet size (( *)P=1.65E-13) relative to the control antibody treated group. Neutralization of IL-1beta also significantly reduced serum amyloid A (SAA) which is an indicator of inflammation-induced acute phase response (( *)P=0.024). While there was no improvement of obesity, a significant improvement of glycemic control and of beta cell function is achieved by this pharmacological treatment which may slow/prevent disease progression in Type 2 Diabetes.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Hypertrophic effect of selective beta(1)-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat

Weinvestigated whether selective ₁-adrenoceptorstimulation causes hypertrophic growth on isolated ventricularcardiomyocytes from adult rat. As parameters for the induction ofhypertrophic growth, the increases of [¹⁴C]phenylalanineincorporation, protein and RNA mass, and cell size weredetermined. Isoproterenol (Iso, 10 µM) alone had no growtheffect. In the presence of the ₂-adrenoceptor antagonist ICI-118551 (ICI, 10 µM), Iso caused an increase in[¹⁴C]phenylalanine incorporation, protein and RNA mass,cell volume, and cross-sectional area. We showed for phenylalanineincorporation that the growth effect of Iso+ICI could be antagonized by₁-adrenoceptor blockade with atenolol (10 µM) ormetoprolol (10 µM), indicating that it was caused by selective₁-adrenoceptor stimulation. The growth response toIso+ICI was accompanied by an increase in ornithine decarboxylase (ODC)activity and expression. Inhibition of ODC by the ODC antagonistdifluoromethylornithine (1 mM) attenuated this hypertrophic response,indicating that ODC induction is causally involved. The growth responseto Iso+ICI was found to be cAMP independent but was sensitive togenistein (100 µM) or rapamycin (0.1 µM). The reaction was enhancedin the presence of pertussis toxin (10 µM). We conclude thatselective ₁-adrenoceptor stimulation causes hypertrophicgrowth of ventricular cardiomyocytes by a mechanism that is independentof cAMP but dependent on a tyrosine kinase and ODC.