Molecular properties of the enzymic phytohemagglutinin of mung bean

Mung bean seeds possess a tetrameric galactose-binding protein that displays two types of activities: (a) a hemagglutinin activity, and (b) an alpha-galactosidase activity. This protein can be reversibly converted by pH changes from a tetrameric form, which possesses both enzymic and hemagglutinin activities, to a monomeric form which possesses enzymic activity only. This observation suggests that the enzymic phytohemagglutinin is an aggregated form of a monomeric alpha-galactosidase. The tetrameric alpha-galactosidase has a pH optimum of about pH 7.0, while the monomeric form displays a pH optimum of 5.6. Circular dichroism difference spectra and inhibition studies suggest that aggregation induces conformational changes in the subunits sufficient to alter their enzymatic properties. The possibility of in vivo changes in subunit equilibria, when combined with the accompanying alterations in activity, provides a new concept worthy of consideration with respect to the physiological role of phytohemagglutinins.     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.     

Purification and properties of an alpha-D-galactoside galactohydrolase from the seeds of Trifolium repens (white clover).

Five alpha-D-galactosidases (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) have been identified by chromatography and polyacrylamide-disc-gel electrophoresis in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidase I has been purified to homogeneity with an approx. 2000-fold increase in specific activity. The enzyme was purified by a procedure which included precipitation by dialysis against citrate/phosphate buffer, pH3.5; (NH4)2SO4 precipitation; hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose column chromatography. Each stage of purification was controlled by polyacrylamide-disc-gel electrophoresis; the purified enzyme showed a single protein band that corresponded to the alpha-D-galactosidic activity. The pH optimum was found to be between pH 3.8 and 4.2; the enzyme is highly thermolabile. Hydrolysis of oligosaccharides and galactomannans has been examined, and it has been found that alpha-galactosidase I exhibits two enzymic activities, namely alpha-D-galactoside galactohydrolase and galactosyltransferase. By the polyacrylamide-gel-electrophoresis method of Hendrick & Smith (1968), and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the mol.wt. has been estimated to be 43 000 and 41 000 respectively. These results indicate that alpha-galactosidase I is a monomeric protein and that both enzymic activities associated with the enzyme reside on the same polypeptide chain.     

Catalytic activity of maize leaf phosphoenolpyruvate carboxylase in relation to oligomerization

The relationship between the state of oligomerization and activity of purified maize leaf phosphoenolpyruvate carboxylase using size exclusion high performance liquid chromatography was examined. Maximum activities of 35 to 38 micromoles per minute per milligram protein were found when 100% of the enzyme was in its tetrameric form. The effects of the sulfhydryl group modifiers CuCl₂ and p-chloromercuribenzoate on enzyme inhibition and the state of aggregation of the protein complex were examined. Aggregation of the enzyme is temperature and pH sensitive with low temperature and high pH favoring depolymerization. Stability of the tetrameric form is largely dependent upon histidyl residues, and to some extent this explains the biphasic response of enzyme activity to changes in MgCl₂ concentrations. Modification of the tetramer's histidyl residues by the inhibitor diethylpyrocarbonate (0.125 millimolar) results in its dissociation to the dimeric form and loss of activity. Subsequent treatment with 0.4 molar hydroxylamine results in reassociation to the tetramer and restoration of enzymic activity.     

Legume alpha-Galactosidases Which Have Hemagglutinin Properties

Four legume species (four genera) were examined and found to contain hemagglutinins with properties similar to those which we have previously described for the enzymic hemagglutinin in Vigna radiata. Examination of extracts by gel filtration and ion exchange chromatography showed that an alpha-galactosidase activity exactly co-purified with a hemagglutinin activity in each of the four species. The alpha-galactosidase activities in the four species were virtually identical to each other with respect to substrate and inhibitor specificity as well as kinetic behavior. Additionally the hemagglutinin activities in all four species displayed very similar carbohydrate specificities. The inhibitor specificities displayed by the enzymes and the hemagglutinins were qualitatively and quantitatively very nearly identical to each other. The remarkable similarities of these proteins, both to each other and to the previously described Vigna enzymic hemagglutinin, suggest that each of these plants may contain a homologue from a specific class of enzymic hemagglutinin.     

Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose.

A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.     

Enzymic preparation of protein G-peroxidase conjugates catalysed by transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for site-directed protein coupling via N(epsilon)-(gamma-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross-linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality revealed the coupling of protein G with an average of three peroxidase molecules.     

CRP,immunologically cross-reacting protein of prolyl hydroxylase. Its role in assembly of active prolyl hydroxylase and cellular localization in L-929 fibroblasts

There are two forms of prolyl hydroxylase in L-929 flbroblasts. One is the enzymatically active tetramer having two α- and two β-subunits. The other is monomeric cross-reacting protein which is enzymatically inactive but is structurally related to β-subunit of the enzyme. Cultured L-929 fibroblasts at mid-log phase were labeled by ³H-labeled amino acid mixture and the radioactivity was chased for 24 h while cells were harvested and plated at higher cell densities in cultures. The results indicated that both α-subunit of the tetrameric prolyl hydroxylase and cross-reacting protein were labeled, but the β-subunit of the tetrameric active prolyl hydroxylase was not labeled until the cells were crowded for 24 h. Using immunofluorescent techniques with antibodies directed against pure tetrameric prolyl hydroxylase, capping or patching was observed when the cells were incubated at 37 °C. Also, it was found that phagosomes prepared from L-929 flbroblasts contained about 30% of total enzyme protein as determined immunologically but contained no significant prolyl hydroxylase activities. Labeling cells with ¹²⁵I by lactoperoxidase, cross-reacting protein was labeled but both α- and β-subunits of tetrameric active prolyl hydroxylase were not labeled. The results indicate that cross-reacting protein can be utilized as the precursor of β-subunit by the cells to form tetrameric active prolyl hydroxylase and that cross-reacting protein is found associated with cytoplasmic membranes.     

Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus.

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.     

A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.     

Membrane interactions of mutated forms of the influenza fusion peptide

We have studied a group of fusion peptides of influenza hemagglutinin in which the N-terminal amino acid, Gly (found in the wild-type peptide), has been systematically substituted with Ala, Ser, Val, or Glu. The activity of the intact hemagglutinin protein with these same substitutions has already been reported. As a measure of the extent of modulation of intrinsic membrane curvature by these peptides, we determined their effects on the polymorphic phase transition of dipalmitoleoylphosphatidylethanolamine. The wild-type peptide is the only one that, at pH 5, can substantially decrease the temperature of this transition. This is also the only form in which the intact protein promotes contents mixing in cells. The Ala and Ser mutant hemagglutinins exhibit a hemifusion phenotype, and their fusion peptides have little effect on lipid polymorphism at low pH. The two mutant proteins that are completely fusion inactive are the Val and Glu mutant hemagglutinins. The fusion peptides from these forms significantly increase the polymorphic phase transition temperature at low pH. We find that the effect of the fusion peptides on membrane curvature, as monitored by a shift in the temperature of this polymorphic phase transition, correlates better with the fusogenic activities of the corresponding protein than do measurements of the isotropic (31)P NMR signals or the ability to induce the fusion of liposomes. The inactivity of the hemagglutinin protein with the hydrophobic Val mutation can be explained by the change in the angle of membrane insertion of the helical fusion peptide as measured by polarized FTIR. Thus, the nature of the interactions of the fusion peptides with membranes can, in large part, explain the differences in the fusogenic activity of the intact protein.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

Isolation of the measles virus hemagglutinin protein in a soluble form by protease digestion.

The hemagglutinin (H) glycoprotein was isolated in a soluble form by digesting measles virus particles with an endoproteinase, Asp-N (from a Pseudomonas fragi mutant). Digestion of H with Asp-N brought about glycopeptides in three different forms, depending on the cleaving site: AHD, which has an M(r) of 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which formed a disulfide-linked homodimer with an M(r) of 132,000, and two monomeric digestion products, AHM-1 (with an M(r) of 64,000) and AHM-2 (with an M(r) of 58,000). The susceptibility of the H glycoprotein to the protease depended on the enzyme concentration. AHD was readily formed at a low concentration of Asp-N, while AHM-1 and AHM-2 required higher and even higher protease concentrations, respectively. All of the cleavage products reacted with monoclonal antibodies to various epitopes of the H protein; however, only AHD showed a significant hemagglutinin activity on African green monkey erythrocytes. The hemagglutinin activities of AHM-1 and AHM-2 were restored after a monoclonal antibody lacking the hemagglutination-inhibiting activity was added to the reaction mixture. AHDs purified by size-exclusion high-pressure liquid chromatography had two associating forms; one had an M(r) higher than and the other an M(r) as high as that of a tetramer. The former was associated noncovalently in addition to having two intermolecular disulfide bonds, and the latter was associated covalently with a single intermolecular disulfide bond and was also duplicated through a noncovalent association. In addition, both AHM-1 and AHM-2, having no intermolecular disulfide bond, were in a dimer form. These results suggest that AHM-1 and AHM-2 are monovalent in the hemagglutinin activity, while AHDs are divalent. Comparative analyses of the N termini of these soluble glycopeptides with the sequence of H suggested that the cysteine residue at position 139 was responsible for the intermolecular disulfide bonding between the monomeric H glycoproteins. The cysteine at position 154 was also suggested to participate in the forming of the intermolecular disulfide bond.     

Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

Molecular modelling of miraculin: Structural analyses and functional hypotheses

Miraculin is a plant protein that displays the peculiar property of modifying taste by swiching sour into a sweet taste. Its monomer is flavourless at all pH as well as at high concentration; the dimer form elicits its taste-modifying activity at acidic pH; a tetrameric form is also reported as active. Two histidine residues, located in exposed regions, are the main responsible of miraculin activity, as demonstrated by mutagenesis studies. Since structural data of miraculin are not available, we have predicted its three-dimensional structure and simulated both its dimer and tetramer forms by comparative modelling and molecular docking techniques. Finally, molecular dynamics simulations at different pH conditions have indicated that at acidic pH the dimer assumes a widely open conformation, in agreement with the hypotheses coming from other studies.     

Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-[LDH-like] malate dehydrogenase from the halophilic archaeon Haloarcula marismortui

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot. The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

Visual arrestin activity may be regulated by self-association.

Visual arrestin is the protein responsible for rapid quenching of G-protein-coupled receptor signaling. Arrestin exists as a latent inhibitor which must be 'activated' upon contact with a phosphorylated receptor. X-ray crystal structures of visual arrestin exhibit a tetrameric arrangement wherein an asymmetric dimer with an extensive interface between conformationally different subunits is related to a second asymmetric dimer by a local two-fold rotation axis. To test the biological relevance of this molecular organization in solution, we carried out a sedimentation equilibrium analysis of arrestin at both crystallographic and physiological protein concentrations. While the tetrameric form can exist at the high concentrations used in crystallography experiments, we find that arrestin participates in a monomer/dimer equilibrium at concentrations more likely to be physiologically relevant. Solution interaction analysis of a proteolytically modified, constitutively active form of arrestin shows diminished dimerization. We propose that self-association of arrestin may provide a mechanism for regulation of arrestin activity by (i) ensuring an adequate supply for rapid quenching of the visual signal and (ii) limiting the availability of active monomeric species, thereby preventing inappropriate signal termination.