Ca2+ channel beta3 subunit enhances voltage-dependent relief of G-protein inhibition induced by muscarinic receptor activation and Gbetagamma

The Ca2+ channel beta subunit has been shown to reduce the magnitude of G-protein inhibition of Ca2+ channels. However, neither the specificity of this action to different forms of G-protein inhibition nor the mechanism underlying this reduction in response is known. We have reported previously that coexpression of the Ca2+ channel beta3 subunit causes M2 muscarinic receptor-mediated inhibition of alpha1B Ca2+ currents to become more voltage-dependent. We report here that the beta3 subunit increases the rate of relief of inhibition produced by a depolarizing prepulse and also shifts the voltage dependency of this relief to more hyperpolarized voltages; these effects are likely to be responsible for the reduction of inhibitory response of alpha1B channels to G-protein-mediated inhibition seen after coexpression of the Ca2+ channel beta3 subunit. Additionally, the beta3 subunit alters the rate and voltage dependency of relief of the inhibition produced by coexpressed Gbeta1gamma1, in a manner similar to the changes it produces in relief of M2 receptor-induced inhibition. We conclude that the Ca2+ channel beta3 subunit reduces the magnitude of G-protein inhibition of alpha1B Ca2+ channels by enhancing the rate of dissociation of the G-protein betagamma subunit from the Ca2+ channel alpha1B subunit.     

His64(E7)-->Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation

To develop an assay for hemin dissociation, His64(E7) was replaced by Tyr in sperm whale myoglobin producing a holoprotein with a distinct green color due to an intense absorption band at 600 nm. Val68(E11) was replaced by Phe in the same protein to increase its stability. When excess Tyr64-Val68 apoglobin is mixed with either metmyoglobin or methemoglobin, the solution turns from brown to green, and the absorbance changes can be used to measure complete time courses for hemin dissociation from either holoprotein. This assay has been used to measure rates of hemin dissociation from native metmyoglobin, four myoglobin mutants (Ala64(E7), Ala68(E11), Phe68(E11), and Glu45(CD3)), native methemoglobin, valence hybrid hemoglobins, and two mutant hemoglobins ((alpha(Gly-E7)beta(native))2, and (alpha(native)beta(Gly-E7))2). Two kinetic phases were observed for hemin dissociation from native human hemoglobin at pH 7.0 and 37 degrees C. Valence and mutant hybrid hemoglobins were used to assign the faster phase (k = 7.8 +/- 2.0 h-1) to hemin dissociation from ferric beta subunits and the slower (k = 0.6 +/- 0.15 h-1) to dissociation from alpha subunits. The corresponding rate for wild-type metmyoglobin is 0.007 +/- 0.004 h-1.     

Epiphyseal distraction for bony bridges: a biomechanical and morphologic study

AT(D)PMg induces dissociation of the alpha 3 beta 3 complex of F1-ATPase from a thermophilic Bacillus strain. PS3, into the alpha 1 beta 1 heterodimers [(1991) Biochim. Biophys. Acta 1056, 279-284] but the location of the AT(D)PMg binding site responsible is not known. From the analysis of AT(D)PMg binding properties of the isolated mutant beta subunit, beta(Y341C), and the stability of the alpha 3 beta(Y341C)3 complex in the presence of AT(D)PMg, we conclude that binding of AT(D)PMg to the Tyr-341 site of the beta subunit(s) in the alpha 3 beta 3 complex triggers the dissociation of the alpha 3 beta 3 complex into the alpha 1 beta 1 heterodimers.     

Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1F0 ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the alpha, beta, gamma, epsilon, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na(+)-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and delta subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or Hi+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.     

Characterization of CD44-mediated hyaluronan binding by renal tubular epithelial cells

Sedimentation equilibrium studies of dilute solutions of tryptophan synthase reveal dissociation from the holoenzyme form, alpha 2 beta 2, into mixtures of alpha beta 2, small amounts of beta 2, and alpha as well as the original alpha 2 beta 2 holoenzyme. The holoenzyme form is stabilized by pyridoxal 5'-phosphate. A new sedimentation equilibrium analytical procedure shows the dissociation of the second alpha subunit to be negatively cooperative. The analytical procedure calculates theoretical error profiles with assumed values of the dissociation constant, k, and a cooperativity parameter until a match is made between one of the theoretical profiles and that computed from experimental data. The latter profile is calculated with an experimentally determined k and assumed values of the cooperativity parameter.     

Quaternary structure regulates hemin dissociation from human hemoglobin

Rate constants for hemin dissociation from the alpha and beta subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 degrees C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated alpha and beta chains and for recombinant hemoglobin tetramers stabilized by alpha subunit fusion. The rate constant for hemin dissociation from beta subunits in native hemoglobin increases from 1.5 h-1 in tetramers at high protein concentration to 15 h-1 in dimers at low concentrations. The rate of hemin dissociation from alpha subunits in native hemoglobin is significantly smaller (0.3-0.6 h-1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-alpha subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric alpha and beta chains are much greater, 12 and 40 h-1, respectively, at pH 7, 37 degrees C. Aggregation of monomers to form alpha1beta1 dimers greatly stabilizes bound hemin in alpha chains, decreasing its rate of hemin loss approximately 20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to beta subunits. A significant reduction in the rate of hemin loss from beta subunits does occur after formation of the alpha1beta2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.     

Tryptophan synthase mutations that alter cofactor chemistry lead to mechanism-based inactivation

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.     

Separation and detection of the alpha- and beta-chains of hemoglobin of a single intact red blood cells using capillary electrophoresis/electrospray ionization time-of-flight mass spectrometry

A single intact red blood cell (erythrocyte) was injected into a capillary electrophoresis column, and following in-capillary lysing the alpha- and beta-chains of the hemoglobin (approximately 450 amol) were separated and detected using capillary electrophoresis/electrospray ionization time-of-flight mass spectrometry. The mass specta of the electropherogram peaks of the alpha and beta chains showed identifiable peaks corresponding to multiply protonated and sodiated alpha- and beta-chains of hemoglobin.     

Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.     

The novel type II prolyl 4-hydroxylase is the main enzyme form in chondrocytes and capillary endothelial cells, whereas the type I enzyme predominates in most cells

Procollagen-proline dioxygenase (EC 1.14.11.2), an alpha2beta2 tetramer in vertebrates, plays a central role in the synthesis of all collagens. Recently an isoform of the alpha subunit, the alpha(II) subunit, was characterized in man and mouse and found to form a tetramer with the same beta subunit as the previously known alpha(I) subunit. We report here that the (alpha(I))2beta2 type I tetramer is the main enzyme form in most cell types and tissues and that its contribution to total prolyl 4-hydroxylase activity in cultured cells increases in confluence. Surprisingly, however, the (alpha(II))2beta2 type II enzyme was found to represent at least about 70% of the total prolyl 4-hydroxylase activity in cultured mouse chondrocytes and about 80% in mouse cartilage, the corresponding percentage in mouse bone being about 45% and that in many other mouse tissues about 10% or less. Immunofluorescence studies on samples from a fetal human foot confirmed these data and additionally indicated that the type II enzyme represents the main or only enzyme form in capillary endothelial cells. Thus the type II prolyl 4-hydroxylase is likely to play a major role in the development of cartilages and cartilaginous bones and also of capillaries.     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit

Run-down of L-type Ca2+ channels in CHO cells stably expressing alpha 1c, alpha 1c beta 1a, or alpha 1c beta 1a alpha 2 delta gamma subunits was studied using the patch-clamp technique (single channel recording). The channel activity (NPo) of alpha 1c channels was increased 4- and 8-fold by coexpression with beta 1a and beta 1a alpha 2 delta gamma, respectively. When membranes containing channels composed of different subunits were excised into basic internal solution, the channel activity exhibited run-down, the time-course of which was independent of the subunit composition. The run-down was restored by the application of calpastatin (or calpastatin contained in cytoplasmic P-fraction) + H-fraction (a high molecular mass fraction of bovine cardiac cytoplasm) + 3 mM ATP, which has been shown to reverse the run-down in native Ca2+ channels in the guinea-pig heart. The restoration level was 64.7, 63.5, and 66.4% for channels composed of alpha 1c, alpha 1c beta 1a, and alpha 1c beta 1a alpha 2 delta gamma, respectively, and was thus also independent of the subunit composition. We conclude that run-down of L-type Ca2+ channels occurs via the alpha 1 subunit and that the cytoplasmic factors maintaining Ca2+ channel activity act on the alpha 1 subunit.     

A reporter mutation approach shows incorporation of the "orphan" subunit beta3 into a functional nicotinic receptor

We have investigated whether the neuronal nicotinic subunit beta3 can participate in the assembly of functional recombinant receptors. Although beta3 is expressed in several areas of the central nervous system, it does not form functional receptors when expressed heterologously together with an alpha or another beta nicotinic subunit. We inserted into the human beta3 subunit a reporter mutation (V273T), which, if incorporated into a functional receptor, would be expected to increase its agonist sensitivity and maximum response to partial agonists. Expressing the mutant beta3(V273T) in Xenopus oocytes together with both the alpha3 and the beta4 subunits resulted in the predicted changes in the properties of the resulting nicotinic receptor when compared with those of alpha3 beta4 receptors. This indicated that some of the receptors incorporated the mutant beta3 subunit, as part of a "triplet" alpha3 beta4 beta3 receptor. The proportion of triplet receptors was dependent on the ratios of the alpha3:beta4:beta3 cRNA injected. We conclude that, like the related alpha5 subunit, the beta3 subunit can form functional receptors only if expressed together with both alpha and beta subunits.     

A hierarchy of disulfide-bonded subunits: the quaternary structure of Eudistylia chlorocruorin

The quaternary structure of the cysteine-rich, approximately 3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at approximately 25 and approximately 33 kDa, and one peak at approximately 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed: L1a-f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (+/-2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/-1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1; D2 at 33 374.1, which had a cysteinylated Cys (a2 + b2 + Cys); and D3 = a1 + b4 at 33 149.4 Da (+/-3.0 Da), with relative intensities D1:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea [Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found to consist only of tetramers T. A model was proposed for the Chl, based on a dimer:tetramer ratio of 2:1: four 206-kDa dodecamers (trimer of tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2 linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (1059 kDa) provide a total mass of 3539 kDa, in good agreement with the 3480 +/- 225 kDa determined previously by STEM mass mapping. The hierarchy of disulfide-bonded globin subunits observed for Eudistylia Chl provides a built-in heterogeneity of hexagonal bilayer structures.     

Specific Cu2+-catalyzed oxidative cleavage of Na,K-ATPase at the extracellular surface

This paper describes specific Cu2+-catalyzed oxidative cleavage of alpha and beta subunits of Na,K-ATPase at the extracellular surface. Incubation of right side-out renal microsomal vesicles with Cu2+ ions, ascorbate, and H2O2 produces two major cleavages of the alpha subunit within the extracellular loop between trans-membrane segments M7 and M8 and L7/8. Minor cleavages are also detected in loops L9/10 and L5/6. In the beta subunit two cleavages are detected, one before the first S-S bridge and the other between the second and third S-S bridges. Na,K-ATPase and Rb+ occlusion are inactivated after incubation with Cu2+/ascorbate/H2O2. These observations are suggestive of a site-specific mechanism involving cleavage of peptide bonds close to a bound Cu2+ ion. This mechanism allows several inferences on subunit interactions and spatial organization. The two cleavage sites in L7/8 of the alpha subunit and two cleavage sites of the beta subunit identify interacting segments of the subunits. L7/8 is also close to L9/10 and to cation occlusion sites. Comparison of the locations of Cu2+-catalyzed cleavages with Fe2+-catalyzed cleavages (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601) suggests division of the membrane sector into two domains comprising M1-M6 and M7-M10/Mbeta, respectively.     

Oxygenation-linked subunit interactions in human hemoglobin: experimental studies on the concentration dependence of oxygenation curves

An experimental study on the concentration dependence of oxygenation curves for human hemoglobin has been carried out between 4 X 10(-8) M heme and 5 X 10(-4) M heme in 0.1 M tris(hydroxymehtyl)aminomethane hydrochloride, 0.1 M NaCl, 1 mM disodium ethylenediaminetetraacetic acid, pH 7.4, 21.5 degrees C. With decreasing hemoglobin concentration the curves show pronounced shifts in position and shape, consistent with dissociation of tetrameric hemoglobin into dimeric species of high affinity and low cooperativity. Combination of these data with independently determined values of dissociation constants for unliganded and fully liganded hemoglobin permits a resolution of the seven parameters necessary to define the linked binding and subunit association processes. The total oxygenation-linked subunit dissociation energy (6.34 kcal) was resolved into intersubunit contact energy changes between alphabeta dimers in tetrameric hemoglobin which accompany binding of the first, middle two, and last oxygen molecules. The resolution is accurate to within approximately +/-0.3 kcal. To within this limit the isolated dimers are found to bind oxygen noncooperatively and with the same affinity as isolated alpha and beta chains. Equally good fits to the data are obtained when dimers are slightly anticooperative. At least three major energetic states are apparently assumed by hemoglobin tetramers, with respect to the alpha1beta2 contact region, corresponding to (a) unliganded, (b) singly liganded, (c) triply and quadruply liganded species. The results do not establish whether these states may be assumed by a single molecule, or whether they arise as averages over a distrubution of conformational states. They do provide unequivocal evidence against a concerted transition at any particular binding step in a system with only two energetic states of tetramer (i.e., an all or none switchover between T and R states at a particular binding step).