Magnesium Binding Sites Research Articles

The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit.

The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence. Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P x pre-tRNA(Asp) complex. The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNA(Asp). Nor does the protein component alter the pH dependence of pre-tRNA(Asp) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state. However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme x pre-tRNA complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P x pre-tRNA complex indirectly by binding and positioning pre-tRNA. Furthermore, RNase P is inhibited by cobalt hexammine (K(I) = 0.11 +/- 0.01 mM) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P. These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.

Structural analysis, identification, and design of calcium-binding sites in proteins.

Assigning proteins with functions based on the 3-D structure requires high-speed techniques to make a systematic survey of protein structures. Calcium regulates many biological systems by binding numerous proteins in different biological environments. Despite the great diversity in the composition of ligand residues and bond angles and lengths of calcium-binding sites, our structural analysis of 11 calcium-binding sites in different classes of proteins has shown that common local structural parameters can be used to identify and design calcium-binding proteins. Natural calcium-binding sites in both EF-hand proteins and non-EF-hand proteins can be described with the smallest deviation from the geometry of an ideal pentagonal bipyramid. Further, two different magnesium-binding sites in parvalbumin and calbindin(D9K) can also be identified using an octahedral geometry. Using the established method, we have designed de novo calcium-binding sites into the scaffold of non-calcium-binding proteins CD2 and Rop. Our results suggest that it is possible to identify calcium- and magnesium-binding sites in proteins and design de novo metal-binding sites.

The Metal-binding Properties of DREAM: EVIDENCE FOR CALCIUM-MEDIATED CHANGES IN DREAM STRUCTURE

DREAM, an EF-hand protein, associates with and modulates the activity of presenilins and Kv4 potassium channels in neural and cardiac tissues and represses prodynorphin and c-fos gene expression by binding to DNA response elements in these genes. Information concerning the metal-binding properties of DREAM and the consequences of metal binding on protein structure are important in understanding how this protein functions in cells. We now show that DREAM binds 1 mol of calcium/mol of protein with relatively high affinity and another 3 mol of calcium with lower affinity. DREAM binds 1 mol of magnesium/mol of protein. DREAM, pre-loaded with 1 mol of calcium, binds 1 mol of magnesium, thus demonstrating that the magnesium-binding site is distinct from the high affinity calcium-binding site. Analysis of metal binding to mutant DREAM protein constructs localizes the high affinity calcium-binding site and the magnesium-binding site to EF-hands 3 or 4. Binding of calcium but not magnesium changes the conformation, stability, and alpha-helical content of DREAM. Calcium, but not magnesium, reduces the affinity of apo-DREAM for specific DNA response elements in the prodynorphin and c-fos genes. We conclude that DREAM binds calcium and magnesium and that calcium, but not magnesium, modulates DREAM structure and function.

Glycogen Synthase Kinase-3 Inhibition by Lithium and Beryllium Suggests the Presence of Two Magnesium Binding Sites

Lithium inhibits (Li+) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg2+), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be2+) is a potent inhibitor of GSK-3 and competes for both Mg2+ and ATP. Be2+ also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li+ and Be2+ on GSK-3, we have devised a novel dual inhibition analysis. When Be2+ and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li+ exerts no interference with ADP inhibition or vice versa. We find, however, that Li+ and Be2+ do interfere with each other. These results suggest that Be2+ competes for two distinct Mg2+ binding sites: one is Li+-sensitive and the other, which is Li+-insensitive, binds the Mg:ATP complex.

X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 Å resolution

The X-ray structure of Mycobacterium tuberculosis TMP kinase at 1.95 Å resolution is described as a binary complex with its natural substrate TMP. Its main features involve: (i) a clear magnesium-binding site; (ii) an alpha-helical conformation for the so-called LID region; and (iii) a high density of positive charges in the active site. There is a network of interactions involving highly conserved side-chains of the protein, the magnesium ion, a sulphate ion mimicking the β phosphate group of ATP and the TMP molecule itself. All these interactions conspire in stabilizing what appears to be the closed form of the enzyme. A complete multialignement of all (32) known sequences of TMP kinases is presented. Subtle differences in the TMP binding site were noted, as compared to the Escherichia coli, yeast and human enzyme structures, which have been reported recently. These differences could be used to design specific inhibitors of this essential enzyme of nucleotide metabolism. Two cases of compensatory mutations were detected in the TMP binding site of eukarotic and prokaryotic enzymes. In addition, an intriguing high value of the electric field is reported in the vicinity of the phosphate group of TMP and the putative binding site of the γ phosphate group of ATP.

Crystal Structure of Activated CheY: COMPARISON WITH OTHER ACTIVATED RECEIVER DOMAINS

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.

Paradoxical Block of Parathormone Secretion Is Mediated by Increased Activity of Gα Subunits

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G alpha subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G alpha subunits of heterotrimeric G-proteins.

An efficient method for solving RNA structures: MAD phasing by replacing magnesium with zinc.

The structure of a 46-nucleotide RNA complex has been successfully solved using multi-wavelength anomalous dispersion (MAD) at the zinc K edge. Taking advantage of the eight magnesium-binding sites, it has been shown that for five of them magnesium could be replaced by zinc. This resulted in an excellent 2.0 A MAD electron-density map. Zinc, in common with some other transition metals, is able to replace magnesium in RNA structures, but zinc has the advantage of its K edge being ideally located at 1.284 A. As most RNA molecules contain magnesium-binding sites, it is suggested that this method could be a valuable alternative to the use of bromo derivatives of bases, which is limited to chemically synthesizable and thus rather short RNA sequences.

Structural and Biochemical Properties Show ARL3-GDP as a Distinct GTP Binding Protein

Background: Based on sequence similarities, Arf-like (ARL) proteins have been assigned to the Arf subfamily of the superfamily of Ras-related GTP binding proteins. They have been identified in several isoforms in a wide variety of species. Their cellular function is unclear, but they are proposed to regulate intracellular transport. Results: The 1.7 Å crystal structure of murine ARL3-GDP provides a first insight into the structural features of this subgroup of Arf proteins. The N-terminal extension of ARL3 folds into an elongated loop region that is hydrophobically anchored onto the surface by burying 1440 Å 2. The features observed suggest that ARL3 releases its N terminus and undergoes a β sheet register shift upon the binding of GTP. The structure and kinetic experiments with fluorescent mGDP demonstrate that tight GDP (but not GTP) binding is achieved in the absence of a magnesium ion. This is due to a lysine residue in the active site, close to the canonical Mg 2+ site found in other GTP binding proteins. This is a distinct feature separating ARL2 and ARL3 from Arf proteins. Conclusion: The disturbed magnesium binding site and the independence of GDP coordination from the presence of Mg 2+ separate ARL2 and ARL3 from Arf proteins. The β sheet register shift, which is similar to that of Arf, that is observed in the present structure, along with the postulated release of the N-terminal extension and the concomitant exposure of a patch of conserved hydrophobic residues in this region suggest that ARL proteins might be localized to target membranes upon exchange of GDP to GTP. Contrary to the situation in Arf, however, the conformational change to ARL-GTP does not require the presence of membranes and might thus be energetically unfavored. Together with the very low affinity described for the interaction of ARL3 with Mg-GTP, this suggests that ARL protein activation requires the presence of effectors stabilizing the GTP coordination rather than guanine nucleotide exchange factors (GEFs).

The crystal structure of yeast phenylalanine tRNA at 2.0 Å resolution: cleavage by Mg2+ in 15-year old crystals

We have re-determined the crystal structure of yeast tRNAPhe to 2.0 Å resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg2+ is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg2+ binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TΨC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg2+.

High resolution refinement of beta-galactosidase in a new crystal form reveals multiple metal-binding sites and provides a structural basis for alpha-complementation.

The unrefined fold of Escherichia coli beta-galactosidase based on a monoclinic crystal form with four independent tetramers has been reported previously. Here, we describe a new, orthorhombic form with one tetramer per asymmetric unit that has permitted refinement of the structure at 1.7 A resolution. This high-resolution analysis has confirmed the original description of the structure and revealed new details. An essential magnesium ion, identified at the active site in the monoclinic crystals, is also seen in the orthorhombic form. Additional putative magnesium binding sites are also seen. Sodium ions are also known to affect catalysis, and five putative binding sites have been identified, one close to the active site. In a crevice on the protein surface, five linked five-membered solvent rings form a partial clathrate-like structure. Some other unusual aspects of the structure include seven apparent cis-peptide bonds, four of which are proline, and several internal salt-bridge networks. Deep solvent-filled channels and tunnels extend across the surface of the molecule and pass through the center of the tetramer. Because of these departures from a compact globular shape, the molecule is not well characterized by prior empirical relationships between the mass and surface area of proteins. The 50 or so residues at the amino terminus have a largely extended conformation and mostly lie across the surface of the protein. At the same time, however, segment 13-21 contributes to a subunit interface, and residues 29-33 pass through a "tunnel" formed by a domain interface. Taken together, the overall arrangement provides a structural basis for the phenomenon of alpha-complementation.

Mutation in the Magnesium Binding Site of hMSH6 Disables the hMutSα Sliding Clamp from Translocating along DNA

In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6. In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion.

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 ( Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54 760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis ( Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme.

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54¿ omitted¿760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.

A loop deletion in the plant acetohydroxy acid isomeroreductase homodimer generates an active monomer with reduced stability and altered magnesium affinity.

Plant acetohydroxy acid isomeroreductase is a stable homodimer which catalyzes in the presence of magnesium an alkyl migration followed by a NADPH-dependent reduction. Since the enzyme exhibits no kinetic cooperativity either for its cofactor (NADPH and magnesium) or for its substrates, the reason for dimerization of this enzyme was not obvious. Recently, crystallographic studies [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] revealed that the loop of residues 422-431 plays a major part in the dimer interface. To understand the role of the quaternary structure of the enzyme, we have deleted residues 423-430 and substituted Phe 431 for serine. This mutant was further overproduced in Escherichia coli, purified to homogeneity, and characterized. Gel filtration and thermodynamic experiments disclosed that this mutant behaves as an active monomer with reduced thermal stability. Furthermore, kinetic and fluorescence experiments showed that the behavior of the monomer with respect to magnesium was greatly altered. These results demonstrate the function of the quaternary structure of plant acetohydroxy acid isomeroreductase in the stabilization of the tertiary structure but also in the stabilization of a high-affinity magnesium binding site.

A enzima delta-aminolevullnato desidratase

Delta-aminolevulinic acid dehydratase (ALA-D, EC 4.2.1.24) is a sulfhydryl-containing enzyme that asymetrically condenses two molecules of delta-aminolevulinic acid (ALA), catalyzing the formation of porphobilinogen, the monopyrrole precursor of ali biological tetrapyrroles (corrins, porphyrins, chlorins). The two ALA molecules have been termed Aside ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. P-side ALA binds first and forms a Schiffbase with an active-site Iysine. ALA-D is a cytosolic enzyme present in mammals, plants, fungi and bacteria. Bovine enzyme has a molecular mass of 280 000 Da and is composed of eight similar subunits of 35 000 Da, but only four of the subunits form a Schiff-base with the substrate (half-site reactivity). ALA-D from all organisms requires a bivalent metal ion for activity. Although the considerable sequence conservation among ALA-D enzyme from various organisms, there are species-dependent differences in metal ion requirements for enzyme activity. ALA-D is a zinc-dependent enzyme in animals, yeast and some bacteria. Mammalian enzyme bounds 8 zinc ions/octamer. Bovine ALA-D contains two types of Zn2+ binding sites (A and B), each at a stoichiometry of four per octamer. A-metal-ion-binding sites, with a single cysteine residue among its ligands, bind the four zinc ions essential for ALA-D activity (catalytic zinc), which plays a role in A-side ALA binding, in inter-ALA bond formation and in product binding. B-metal-ion-binding sites, with four cysteine residues among its ligands, bind zinc ions refered to as structural, which seems to be involved in the protection of sulfhydryl groups from oxidation. An A-zinc-ion-binding site has been proposed to be present at a number of four per octamer on the enzyme from plants, but has not been demonstrated yet. ALA-D from plants contains two types of magnesium binding sites: four B-metal-ion-binding sites (bind magnesium essential for ALA-D activity) and eight C-metal-ion-binding sites (bind magnesium that activates the enzyme but is not essential for activity). The cysteine-rich sequence of mammalian ALA-D that presumably corresponds to the B-metalion-binding site is replaced by an aspartate-rich sequence in plant ALA-D, probably accounting for the difference on metal-ion requirement (Mg2+ instead of Zn2+ on B-metal-ion-binding site from plant ALA-D). E. coli ALA-D binds eight Zn2+ (presumably four at A-metal-ion-binding site and four at B-metalion-binding site) and eight Mg2+ (presumably at C-metal-ion-binding site) per octamer. Due to its sulfhydrilic nature ALA-D is inhibited by heavy metals such as lead and mercury, serving as a measure of metal intoxication. In addition the inhibition of this enzyme has been implicated with pathological changes observed in some types of porphyrias, hepatorenal tyrosinemia and after lead or mercury exposure. ALA-D inhibition may impair haeme biosynthesis and leads to ALA accumulation, which besides being a potent agonist of y-aminobutiric acid autoreceptors may act as a prooxidant.

Interactions between two divalent ion binding sites in N-methyl-D-aspartate receptor channels.

N-methyl-D-aspartate receptor channels exhibit a high permeability for calcium ions. In this report, we confirm that calcium ions permeate effectively through the wild-type channels, and find that their presence within the pore blocks the flux of sodium and other ions. Further proof for this ionic block comes from the analysis of the epsilon 1(N614Q) mutation where the high permeability of calcium is unchanged but the block by calcium ions is increased twofold. In both the wild-type and mutant channels, calcium ion block is independent of membrane voltage; therefore, the calcium binding site is outside the voltage gradient through the pore and must be close to the extracellular mouth of the ion conductance pathway. This calcium site is distinct from the magnesium binding site, which lies 80% into the pore's electrostatic field and thus exhibits a marked voltage dependence of binding. The epsilon 1(N614Q) mutation reduces the affinity of magnesium ion for its binding site but increases the affinity of calcium ion for its binding site. Since a single mutation perturbs two distinct binding sites in opposite ways, we postulate that binding of divalent ions at the two sites interact.

Metal-binding sites in the major groove of a large ribozyme domain

Background Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific three-dimensional structures that confer activity. The 2.8 å crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4–P6 domain, provides the first detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold can now be examined in detail. Results Heavy-atom derivatives used for the structure determination reveal characteristics of some of the metal-binding sites in the P4–P6 domain. Although long-range RNA–RNA contacts within the molecule primarily involve the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by G.U wobble base pairs. In the native RNA, two of the sites are occupied by fully-hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain. Conclusions Bound at specific sites in the P4–P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G.U base pairs in ribosomal RNAs are magnesium-binding sites.

Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties

Human recombinant α-parvalbumin (PV wt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca 2+ binding in the CD Ca 2+-binding site (PV E62V, PV D51A, PV D51A,62V), the EF site (PV E101V, PV D90A, PV D90A,E101V) or in both (PV E62V,E101V, PV D51A,D90A, Pv D51A,E62V,D90A,EIO1V), were expressed and purified. Flow dialysis revealed that PV wt binds 2 Ca 2+ with equal K′ Ca, of 2.3 × 10 7M −1 and that Mg 2+ competes with a K′ Mg.compet.of 4.9 × 10 3M −1. The three mutants with an inactivated CD site bind 1 Ca 2+ with K′ Ca, of 2.0 to 2.3 × 10 7M −1and K′ Mg.compet.of 3.4 to 4.6 × 10 3M −1, i.e. very similar to those of PV wt. The mutants with an inactivated EF site bind 1 Ca 2+ with K′ Ca, values of 7.9 × 10 6, 4.5 × 10 6and 3.6 × 10 6M −1, for PV D91A, PV E102V and PV E101V,D91A, respectively. The K′ Mg.compet. values of these mutants are about 4-times lower than in PV wt. The three mutants with both sites inactivated bind neither Ca 2+ nor Mg 2+. After excitation at 259 nm, human PV wt which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca 2+ or Mg 2+ to PV wt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca 2+ or Mg 2+. Specific antibodies against human α-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated.

Modification of the N-methyl- d-aspartate (NMDA) receptor in the brain of newborn piglets following hyperventilation induced ischemia

The present study tests the hypothesis that cerebral ischemia induced by severe hypocapnia modifies the N-methyl- d-aspartate (NMDA) receptor/ion channel complex in the cerebral cortical cell membranes of newborn piglets. Studies were performed in six newborn piglets subjected to ischemic hypoxia induced by hyperventilation (PaCO 2, 9–11 mmHg) for 1 h. Comparisons were made to a normoxic group on room air ( n = 6). Following hyperventilation, phosphocreatine decreased 80%, but ATP remained unchanged. NMDA receptor activation was determined by measuring [ 3H]MK-801 binding at concentrations varying from 2.5 to 50 nM. Following hyperventilation, B max decreased 52% to 0.50±0.04 pmol/mg protein ( P = 0.001); however, the K d value was unchanged at 7.45 ± 0.79 nM. Spermine and magnesium dependent activation of the NMDA receptor was determined in the hyperventilated and control groups. With spermine concentrations increasing from 2.5 to 50 μM the maximal spermine dependent activation in the normoxic group was 13.7±7.93% which occurred at a concentration of 3.75±1.37 μM. In the hyperventilated group maximal activation was 32.4±23.5% ( P = 0.095) at 4.58±2.46 μM ( P = ns). With magnesium concentrations increasing from 2.5 to 100 μM the maximal magnesium dependent activation in the normoxic group was 17.0±13.6% which occurred at a concentration of 22.5±6.12 μM. In the hyperventilated group maximal activation was 26.3 ± 14.9% ( P = ns) at 4.58 ± 2.92 μM ( P < 0.0001). These data show that with less severe tissue hypoxia, as evidenced by conservation of ATP, there is less modification of the NMDA receptors. Ischemia induced by hyperventilation leads to an increase in spermine activation of the NMDA receptor, and the NMDA receptor is much more sensitive to magnesium as evidenced by the maximal activation occurring at a significantly lower magnesium concentration. Ischemia induced by hyperventilation modifies the spermine, magnesium, and MK-801 binding sites of the NMDA receptor and may result in increased NMDA receptor mediated neurotoxicity in the newborn brain.