Biomedical subjects

C R Goward

Publications and source records attributed to C R Goward.

At least 19 recordsLinked to original sources

PubMed1997

Crystallization of cytochrome b562 from Erwinia chrysanthemi.

Cytochrome b(562) from Erwinia chrysanthemi has been crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. X-ray precession photographs show that the crystals formed belong to either of the enantiomorphic space groups P4(1)2(1)2 or P4(3)2(1)2 with the cell parameters a = b = 98.6 and c = 62.7 A. Estimation of the crystal density and consideration of the possible values for V(m) indicate that there is either a dimer or trimer in the asymmetric unit. Experiments using the synchrotron radiation source at the CCLRC Daresbury Laboratory have shown that the crystals diffract to at least 2.7 A resolution. An analysis of the N-terminal sequence indicates that this cytochrome shows limited homology to the cytochrome b(562) from E. coli. Determination of the structure will therefore allow analysis of the relationship between these two proteins.

Journal Article↗

PubMed1994

A single amino acid mutation enhances the thermal stability of Escherichia coli malate dehydrogenase.

The stability of wild-type Escherichia coli malate dehydrogenase was compared with a mutant form of the enzyme with the amino acid residue at position 102 changed from arginine to glutamine. The mutation occurs on the underside of a mobile loop which closes over the active-site cleft on formation of the enzyme/cofactor/substrate ternary complex. The mutant enzyme is kinetically compromised while the wild-type enzyme is highly specific for oxaloacetate. The mutant enzyme was shown to be more resistant to irreversible thermal denaturation by thermal inactivation experiments and high-sensitivity differential scanning calorimetry than the wild-type enzyme. In contrast, resistance of both enzymes to reversible unfolding in guanidinium chloride was similar. Circular dichroic spectropolarimetry shows the secondary structures of the enzymes are similar but there is a demonstrable difference in tertiary structure. From the position of the mutation, it is conjectured that the substitution on a mobile surface loop results in partial closure of the loop and greater resistance to thermal inactivation of the mutant enzyme. However, molecular modelling combined with circular dichroic spectropolarimetry indicate that the mutation may have a more widespread effect on the structure than simply partial closure of the mobile surface loop as the environment of distant tyrosine residues is altered. Resistance of the wild-type enzyme to thermal inactivation can be increased by cofactor addition, which may have the effect of partial closure of the mobile surface loop, but has little effect on the mutant enzyme.

Arginine↗

PubMed1994

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.

Amino Acid Sequence↗

PubMed1994

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase.

The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

Amino Acid Sequence↗

PubMed1994

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.

Amino Acid Sequence↗

PubMed1993

Preparation of DNA polymerase from Bacillus caldotenax.

A procedure with four chromatography steps was developed for the purification of DNA polymerase from Bacillus caldotenax by using fast protein liquid chromatography. The procedure was suitable for use with process-scale media. Elution profiles obtained from ion-exchange chromatography and triazine-dye affinity chromatography with fast protein liquid chromatography and process-scale media were similar. The enzyme showed stronger interaction, however, with phenyl-Sepharose FF in the scaled-up process than with the phenyl-Superose used in fast protein liquid chromatography. The surprising binding of the DNA polymerase to sulphonated ion-exchange media at pH 7.5 may be explained by the structure of the enzyme.

Bacillus↗

PubMed1993

Molecular evolution of bacterial cell-surface proteins.

The cell-surface proteins of the infective bacteria Streptococcus and Staphylococcus are probably involved in the process of infection. These proteins share many features including secretion signal peptides, cell-wall spanning regions, membrane anchor domains and repeated domains of various functions. These common features may have evolved by gene duplication and swapping of gene fragments.

Amino Acid Sequence↗

PubMed1992

The importance of arginine 102 for the substrate specificity of Escherichia coli malate dehydrogenase.

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

Amino Acid Sequence↗

PubMed1992

Purification and properties of DNA polymerase from Bacillus caldotenax.

A thermostable DNA polymerase was prepared from Bacillus caldotenax by using a four-step chromatography procedure. The protein exists as a monomer of M(r) 94,000, has a pI of 4.9 and has no associated 3'-5' or 5'-3'-exonuclease activities or endonuclease activity. The temperature optimum of the enzyme was about 70 degrees C and the pH for maximum activity was about 7.5. The enzyme has an absolute requirement for a bivalent cation, and maximum activity was obtained at the unusually high concentration of 70 mM-MgCl2. Mg2+ could be replaced by MnCl2 or CoCl2, with decreased activity, at the lower optimal concentrations of 1 mM and 2.5 mM respectively. Enzyme activity was inhibited in the presence of 2',3'-dideoxy-TTP, arabinosyl-CTP and aphidicolin. Enzyme activity was stimulated with KCl concentrations of about 100 mM, and concentrations of univalent salts above about 150 mM inhibited activity. The enzyme could use activated calf thymus DNA, poly(dA).p(dT)10 or primed single-stranded phage M13 DNA as a template and maximum activity was obtained with poly(dA).p(dT)10. The enzyme was inactive on unprimed single-stranded DNA, double-stranded DNA and polyribonucleotide template/primer. The apparent Km values for individual dNTPs, determined with the other dNTPs at saturating concentrations, were 5.7 microM (dCTP), 6.3 microM (dATP, dGTP) and 6.4 microM (dTTP). The Km value for the overall incorporation of each dNTP from an equimolar mixture of all four dNTPs was 24.7 microM. The kcat. value was about 1.05 s-1. The kcat./Km value was 0.16-0.18 M-1.s-1 for individual dNTPs and 0.04 for the incorporation of an equimolar mixture of all four dNTPs. Some of the properties of the enzyme show it may be classified as an alpha-Type DNA polymerase.

Animals↗

PubMed1992

Rapid large-scale preparation of recombinant Erwinia chrysanthemi L-asparaginase.

L-asparaginase from Erwinia provides an alternative to the enzyme from E. coli for the effective treatment of acute lymphoblastic leukaemia. A procedure was required for the large-scale partial purification of the recombinant Erwinia enzyme cloned and expressed in Erwinia. Enzyme was extracted from Erwinia at high pH and extraneous protein precipitated at low pH. S-Sepharose FF was selected as the medium of choice for the chromatography step since it was adequate for the high flow rates required (linear flow rate 315 cm h-1) and the methylsulphonate functional groups exploited the high pI of the enzyme by allowing binding of L-asparaginase at pH 4.8 while most of the other proteins passed through the column. The useful capacity of the matrix was up to 34 mg enzyme/ml matrix at a linear flow rate of 95 cm h-1 and 15.4 mg enzyme/ml matrix at a linear flow rate of 315 cm h-1. Weakly bound protein was removed by a wash at pH 6.0. The L-asparaginase was eluted by a wash at pH 6.8 (linear flow rate 95 cm h-1) and was substantially pure, only requiring polishing steps to be suitable for use as a parenteral agent. The purity of the protein was complemented by a 92% recovery of active enzyme from this cation-exchange matrix.

Asparaginase↗

PubMed1992

Amplified expression and large-scale purification of protein G'.

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.

Amino Acid Sequence↗

PubMed1991

Sequential 1H NMR assignments and secondary structure of an IgG-binding domain from protein G.

Protein G is a member of a class of cell surface bacterial proteins from Streptococcus that bind IgG with high affinity. A fragment of molecular mass 6988, which retains IgG-binding activity, has been generated by proteolytic digestion and analyzed by 1H NMR. Two-dimensional DQF-COSY, TOCSY, and NOESY spectra have been employed to assign the 1H NMR spectrum of the peptide. Elements of regular secondary structure have been identified by using nuclear Overhauser enhancement, coupling constant, and amide proton exchange data. The secondary structure consists of a central alpha-helix (Ala28-Val44), flanked by two portions of beta-sheet (Val5-Val26 and Asp45-Lys62). This is a fundamentally different arrangement of secondary structure from that of protein A, which is made up of three consecutive alpha-helices in free solution (Torigoe et al., 1990). We conclude that the molecular mechanisms underlying the association of protein A and protein G with IgG are different.

Amino Acid Sequence↗

PubMed1991

The secondary structure of protein G', a robust molecule.

The secondary structure of recombinant streptococcal Protein G' was predicted and compared with spectropolarimetric data. The predicted secondary structure consisted of 37 +/- 4% alpha-helix and 30 +/- 5% beta-sheet, whereas the values obtained from c.d. data were 29 +/- 2% alpha-helix and 41 +/- 3% beta-sheet. An alpha-helix-beta-sheet/turn-alpha-helix motif is conjectured to comprise the Fc-binding unit. The c.d. spectra in the near u.v. and far u.v. show that the Protein G' molecule is stable to heating at 100 degrees C and to extremes of pH (pH 1.5 to 11.0). The protein retained biological activity at these extremes. The molecule uncoils above pH 11.5 in a time-dependent fashion. Unfolding of the molecule in guanidinium chloride was monitored by c.d. and fluorescence emission; 3 M-guanidinium chloride was required to unfold the protein by 50%. The protein was completely unfolded in 5.5 M-guanidinium chloride and fully refolded with restoration of activity after removal of guanidinium chloride.

Amino Acid Sequence↗

PubMed1990

Expression and purification of a truncated recombinant streptococcal protein G.

The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000.

Amino Acid Sequence↗

PubMed1990

Colorimetric glucose assay using thermostable glucokinase.

A method for assaying glucose in serum or plasma samples using a thermostable glucokinase was developed. Glucokinase from Bacillus Stearothermophilus was coupled with glucose-6-phosphate dehydrogenase to produce NADPH, which reduced the tetrazolium dye MTT to its formazan. Detection of the product at 660 nm allowed samples containing up to 30 mmol/L glucose to be assayed with an endpoint method. Use of the optimal wavelength for formazan detection, 570 nm, increased sensitivity for NADPH detection by over threefold compared to UV detection. The stability of glucokinase assay mixtures was extensively studied, with variation in buffers, salt and enzyme stabilizers. Maximal half life for reagent stability at room temperature was approximately 30 days, with storage of assay mixtures in two solutions. Various drugs and metabolites were tested for interference in the method and no significant interferences were found.

Blood Glucose↗

PubMed1989

Solubilization of IgG-binding proteins from group A and G streptococci.

The release of IgG-binding proteins from the cell surface of streptococcal strains AR-1 and G148 with various proteolytic enzymes, acid, alkali or SDS was investigated. The IgG-binding proteins were purified by affinity chromatography using IgG-Sepharose Fast Flow. After SDS-polyacrylamide gel electrophoresis and immuno-electroblotting the major proteins identified varied in relative molecular mass from 15,000 to 65,000 depending on the solubilizing agent used. The results showed that solubilization with trypsin gave the highest yield of IgG-binding proteins, that strain G148 yielded about twice the amount of protein as strain AR-1, and that elastase released an IgG-binding protein of high relative molecular mass of 65,000.

Bacterial Proteins↗

PubMed1988

Large-scale purification of the chromosomal beta-lactamase from Enterobacter cloacae P99.

Homogeneous beta-lactamase (beta-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the beta-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.

Anti-Bacterial Agents↗

PubMed1987

The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.

Binding Sites↗