Biomedical subjects

J E Churchich

Publications and source records attributed to J E Churchich.

At least 19 recordsLinked to original sources

PubMed1997

Conformational changes at the nucleotide binding of GroEL induced by binding of protein substrates. Luminescence studies.

2'-Deoxy-3'-anthraniloyl adenosine-5-triphosphate (ANT-dATP) coordinated to Tb3+ was used as an environmentally sensitive probe of the nucleotide-binding site of GroEL. Tb3+.ANT-dATP recognizes the nucleotide-binding site of GroEL and inhibits ATPase activity. Sensitized luminescence, arising from resonance energy transfer from the anthraniloyl moiety to Tb3+, is substantially enhanced in the presence of GroEL. Binding of denatured mitochondrial malate dehydrogenase to the apical domain of GroEL causes a red shift in the fluorescence emitted by anthraniloyl and further enhancement in the phosphorescence emitted by Tb3+ upon excitation at 320 nm. It is suggested that binding of the protein substrate initiates domain movement, which is extended to the nucleotide-binding site. The luminescence results are discussed in reference to the structure of GroEL derived from x-ray crystallographic studies.

Adenosine Triphosphate↗

PubMed1997

Activation of partially folded mitochondrial malate dehydrogenase by thioredoxin.

CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenase is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.

Anilino Naphthalenesulfonates↗

PubMed1997

Recombinant brain 4-aminobutyrate aminotransferases overexpression, purification, and identification of Lys-330 at the active site.

4-Aminobutyrate aminotransferase (4-aminobutyrate: 2-oxoglutarate aminotransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid shunt. It catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde. In an effort to clarify the structure-function relationships of 4-aminobutyrate aminotransferase, we analyzed 4-aminobutyrate aminotransferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4-aminobutyrate aminotransferase was overexpressed in Escherichia coli. The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catalytic activity. The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5'-P. To verify the catalytic site of 4-aminobutyrate aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis. Overexpression and purification of the mutated 4-aminobutyrate aminotransferase (K330R) were performed by the same method used the purification of wild-type 4-aminobutyrate aminotransferase. The purified and renatured K330R protein did not show the catalytic activity of wild type 4-aminobutyrate aminotransferase. Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5'-P covalently linked to the protein. From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the aminotransferase.

4-Aminobutyrate Transaminase↗

PubMed1997

Binding of the activating ion Co(II) to myo-inositol monophosphatase monitored by fluorescence and phosphorescence spectroscopy.

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 microM, whereas the apparent activation constant Ka is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 A of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 microM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.

Animals↗

PubMed1996

Reversible denaturation of myo-inositol monophosphatase. The stability of the metal-binding loop.

The unfolding of bovine brain myo-inositol monophosphatase by guanidine. HCl (Gdn. HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn.HCl-treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn.HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal-binding loop. Denaturation of the enzyme by Gdn.HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn.HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1 x 10(6)M-1. It is suggested that a residual structure of denatured myo-inositol monophosphatase is responsible for the binding of terbium ions. The kinetics of unfolding and refolding as a function of Gdn.HCl concentration were monitored by protein fluorescence in a stopped-flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80-65 s-1 at 25 degrees C. The refolding kinetics fit monoexponential functions with rate constants in the range 120-65 s-1 depending on the Gdn.HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.

Animals↗

PubMed1996

Spectroscopic studies of myo-inositol monophosphatase with a novel fluorescent substrate.

myo-Inositol monophosphatase catalyzes dephosphorylation of the synthetic substrate anthraniloyl-2'-AMP. Binding of this fluorescent substrate to Tb(III)-monophosphatase was monitored by luminescence spectroscopy. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor anthraniloyl moiety and the acceptor Tb(III) was calculated to be R = 10 angstroms. The binding studies support the earlier observation of Bone et al. (Proc. Natl. Acad. Sci. USA 89 (1992) 10031-10035) that the substrate and the lanthanide Gd(III) interact with a common binding domain of the protein. The catalytic activity of the monophosphatase is completely dependent upon Mg(II) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.

Adenosine Monophosphate↗

PubMed1996

Structural changes of beta-lactoglobulin B induced by urea. Evidence of residual structure.

Several spectroscopic methods have been used to study the structure of beta-lactoglobulin B at pH 2.1 in the presence of 8M urea. Fluorescence and polarization of fluorescence spectroscopy measurements indicate that the two tryptophanyl residues of the protein are exposed to the solvent in the denatured state. CD in the far-UV indicates that the amount of secondary structure in the denatured state is comparable to that found in the native state, whereas the CD spectrum in the near-UV shows that the tertiary structure is not completely disordered. The results of one-dimensional 1H NMR spectroscopy show that some local non-random structure is maintained in the denatured state, but most of the polypeptide chain has an extended non-globular conformation under the conditions of the present experiments. This conclusion is reinforced by the results of two-dimensional 1H NMR conducted on denatured samples of beta-lactoglobulin B. The study of states with intermediate levels of order will aid the understanding of how the native structure of beta-lactoglobulin B is organised during the refolding pathways.

Animals↗

PubMed1995

Binding of a fluorescent nucleotide analog to Hsc70. The effect of peptide protein interactions on the luminescence properties of the probe.

2'-Deoxy-3'-anthraniloyladenosine-5-triphosphate(Ant-dATP) was used as an environmentally sensitive probe of the nucleotide-binding site of the molecular chaperone Hsc70. When coordinated to the lanthanide ion Tb3+, Ant-dATP is not hydrolyzed by Hsc70. The lanthanide ion acts as strong competitive inhibitor with respect to Mg2+ (Ki = 0.1 microM). Tb.Ant-dATP recognizes the nucleotide site of Hsc70 as revealed by an increase in the emission anisotropy from 0.03 to 0.21 and by a change in the fluorescence-decay time from 2.52 ns to 3.75 ns. Sensitized luminescence arising from resonance energy transfer from the anthraniloyl group to Tb3+ is substantially enhanced in the presence of Hsc70. Binding of a 20-amino-acid-residue peptide (Rnase-S peptide) to Hsc70 causes a blue shift in the fluorescence spectrum of Ant-dATP and enhances Tb3+ luminescence upon excitation at 330 nm. It is postulated that binding of the peptide to the COOH-terminal domain of Hsc70 initiates domain movement and the structural changes might extend to the nucleotide-binding site.

Adenosine Triphosphate↗

PubMed1995

Screening and sequence determination of a cDNA encoding the human brain 4-aminobutyrate aminotransferase.

A human brain cDNA library constructed in the lambda ZAP II vector was screened using a fragment of pig brain cDNA encoding 4-aminobutyrate aminotransferase (pGaba-t). A cDNA that encodes the human brain Gaba-t (hGaba-t) has been isolated from the library and sequenced. Using the GenBank and EMBL databases, comparison of the predicted amino-acid sequence of hGaba-t with the pig enzyme revealed 95.4% homology.

4-Aminobutyrate Transaminase↗

PubMed1995

Characterization of monomeric 4-aminobutyrate aminotransferase at low pH.

4-Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time-dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, phi obs = 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL approximately 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped-flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped-flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4-aminobutyrate aminotransferase in the cell.

4-Aminobutyrate Transaminase↗

PubMed1994

Unfolding of 4-aminobutyrate aminotransferase equilibrium and kinetic studies.

The unfolding of pig liver 4-aminobutyrate aminotransferase by urea has been investigated at equilibrium. The overall process was reversible as judged from the recovery of catalytic activity after dilution of urea-treated samples. Unfolding of the enzyme was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the fluorescence and CD changes between 2 and 8 M urea, and the lack of any discernible plateau suggests that unfolding of the protein is a cooperative process. The unfolding of 4-aminobutyrate aminotransferase as a function of urea concentration was monitored by fluorescence measurements of the tryptophanyl residues. The kinetic results indicate that the aminotransferase unfolds in a single kinetic phase. Unfolded 4-aminobutyrate aminotransferase recovers immediately its catalytic activity upon dilution with buffers of neutral pH. Based on equilibrium and kinetic results, a two-state model for the unfolding of the aminotransferase is proposed.

4-Aminobutyrate Transaminase↗

PubMed1994

The binding of substrates and inhibitors to the metal center of myoinositol monophosphatase.

The synthetic substrate anthraniloyl-beta-glycerol-P binds to myoinositol monophosphatase with a Kd = 5 microM at pH 7.5. The anthraniloyl chromophore, excited at 330 nm, sensitizes the long lived luminescence of bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor-acceptor pair was calculated to be R = 10 A. Tb(III) binds to the monophosphatase with a Kd = 2 microM, whereas Ca-(II) displaces the lanthanide at concentrations above 0.1 mM. The binding studies support the notion that Tb(III), Ca(II) and Mg(II) interact with a common binding site on the protein. Phosphate ion, a strong competitive inhibitor, perturbs the luminescence of bound Tb(III), whereas the substrate beta-glycero-P has no effect on the luminescence yield and long-lived emission of bound Tb(III). It is suggested that the phosphate group of the substrate is not in direct contact with the metal ion coordinated to several amino acid residues of the enzyme.

Aminobenzoates↗

PubMed1994

Luminescence spectroscopy of pyridoxic acid and pyridoxic acid bound to proteins.

Luminescence techniques, i.e. fluorescence and phosphorescence, have been employed to study pyridoxic acid bound to proteins through a stable amide linkage. Proteins tagged with 4-pyridoxic acid display the following fluorescence properties: (a) emission and excitation spectra centered at around 430 and 320 nm, respectively; (b) fluorescence quantum yields of 0.3-0.4 and (c) average decay times covering the range 8-9.6 ns. The fluorescence properties of the probe have been used to study the dynamics of the protein in the nanosecond time scale. In the absence of molecular oxygen, free and bound 4-pyridoxic acid exhibit long-lived emission at room temperature. The long-lived emission is red-shifted when compared to fluorescence and decays with average life times ranging over 2.2-0.6 ms depending on the nature of the protein. The fluorophore pyridoxic acid covalently linked to proteins is suitable to study the dynamics of proteins, i.e. fast and slow motions of the macromolecule in the nanosecond and millisecond time scales, respectively.

Fluorescence Polarization↗

PubMed1994

Interaction of 70-kDA heat shock cognate protein with peptides and myo-inositol monophosphatase.

Fluorescence techniques have been used to investigate the interaction of bovine 70-kDa heat shock cognate protein (Hsc 70) with small molecular weight peptides and myo-inositol monophosphatase. The emission properties of Hsc 70 remain invariant upon addition of ATP. The results of steady-state fluorescence indicate that the tryptophan residues of Hsc 70 are exposed to the rapidly relaxing aqueous solvent. Binding of residues 1-20 of ribonuclease A (RNase S-peptide) to Hsc 70 causes protein fluorescence quenching which was used to determine a dissociation constant Kd = 2.7 microM for the binary Hsc 70.RNase S-peptide complex. The octapeptide corresponding to the NH2-terminal portion of sickle cell hemoglobin recognizes Hsc 70 and binds with a Kd = 9.3 microM. Binding of RNase S-peptide to Hsc 70 produces a small enhancement of ATPase activity. Unfolded myo-inositol monophosphatase, tagged with the fluorescent probe 5-[2-(2-iodoacetamido)ethylamino]-1-naphthalenesulfonic acid, recognizes Hsc 70; the formation of a stable complex was detected by steady-state emission anisotropy measurements. The rate and extent of recovery of catalytic activity of unfolded myo-inositol monophosphatase is not influenced by Hsc 70. It is suggested that interaction of Hsc 70 with unfolded proteins in the cell may be able to delay the formation of misfolded structures.

Adenosine Triphosphatases↗

PubMed1994

Myo-inositol monophosphatase: binding of terbium and a cross-linking reagent to the catalytic site cavity.

In the presence of myo-inositol monophosphatase, terbium ions can be excited by energy transfer from the aromatic side chains of the protein. This enhancement of Tb3+ luminescence due to its binding to the enzyme at pH 6.5 was used to determine the dissociation constant (56 microM) for the monophosphatase-Tb3+ complex. Competition luminescence studies also indicated that calcium ions could compete with terbium ions for binding to the enzyme with a dissociation constant of 200 microM. Conditions were determined in which approximately 1 mol of o-phthaldehyde reacts with one subunit of the dimeric protein, yielding a stable fluorescent isoindole derivative. The kinetics of the reaction, monitored by fluorescence spectroscopy, yields a second-order rate constant (K2 = 110 M-1 s-1). The inactivation of the monophosphatase by o-phthaldehyde has been shown to be protected by the substrate beta-glycerolphosphate. Essentially, no formation of any isoindole derivative is detected after one sulfhydryl residue of the enzyme is reacted with N-ethylmaleimide. It is suggested that the site of the isoindole chromophore formed upon reaction of the enzyme with o-phthaldehyde includes one lysyl and one cysteinyl residue located in the cavity of the catalytic site.

Animals↗

PubMed1994

Brain myo-inositol monophosphatase: activity of the single subunit in a dimeric enzyme.

Reversible dissociation of the dimeric structure of brain myo-inositol monophosphatase into subunits was attained by the addition of guanidine-HCl (4M). The molecular mass of the subunits (29 KDa) was determined by HPLC chromatography. Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is determined to be catalytically active, indicating that monomeric subunit of the phosphate is capable of conducting independent catalysis.

Animals↗

PubMed1993

Reversible unfolding of pyridoxal kinase.

The unfolding of brain pyridoxal kinase by guanidinium HCl has been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after removal of guanidinium HCl. Unfolding of pyridoxal kinase was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the spectroscopic changes between 0.2 and 1.5 M guanidinium HCl, and the lack of any discernible plateau suggests that unfolding of the monomer is a cooperative process. A compact intermediate on the unfolding pathway of pyridoxal kinase could not be detected by the method of denaturant gel filtration. The fluorescent analogs of the substrates ATP and pyridoxal were used to assess differences in stability among the domains of the protein. Based on fluorescence and steady emission anisotropy results, it is postulated that the nucleotide domain is more stable than the pyridoxal domain of the kinase.

Animals↗

PubMed1993

Isolation and characterization of recombinant mitochondrial 4-aminobutyrate aminotransferase.

4-Aminobutyrate aminotransferase, which catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde, is a key enzyme of the 4-aminobutyrate shunt. The amino acid sequence predicted from the cDNA sequence shows that the precursor protein consists of the mature enzyme of 473 amino acid residues and an amino-terminal segment of 27 amino acids (Kwon, O. S., Park, J., and Churchich, J. E. (1992) J. Biol. Chem. 267, 7215-7216). A recombinant 4-aminobutyrate aminotransferase has been expressed in Escherichia coli using pETIId as expression vector. The protein has been purified and characterized as a dimer (2 x 55 kDa). NH2-terminal sequence analysis has revealed the presence of an extra amino-terminal segment (signal peptide) predicted from the cDNA sequence. The isolated precursor of 4-aminobutyrate aminotransferase contains pyridoxal 5-phosphate and exhibits catalytic activity (18 units/mg) comparable to that of the mature enzyme (20 units/mg). The presequence peptide of the precursor of mitochondrial 4-aminobutyrate aminotransferase does not interfere with the folding and functional properties of the mature moiety of the aminotransferase.

4-Aminobutyrate Transaminase↗