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J E Coleman

Publications and source records attributed to J E Coleman.

At least 37 records · Page 2Linked to original sources
PubMed1996

A zinc-binding domain involved in the dimerization of RAG1.

Recombination-activating gene 1 (RAG1), as well as RAG2, are the only lymphoid-specific genes required for V(D)J recombination. RAG1 protein contains a C3HC4 zinc-binding motif (zinc ring finger) that binds two zinc ions. We have found that RAG1 contains additional zinc-binding motifs in the form of two separate C2H2 zinc finger sequences. One of the zinc fingers, in combination with the C3HC4 subdomain, forms a highly specific dimerization domain. A combination of biophysical techniques has been used to determine the energetics of association, the overall shape of the dimerization domain, and the relative orientation of the monomeric subunits within the dimer. These results provide direct evidence that a C3HC4 motif is involved in a protein-protein interaction, in this case via homodimer formation. In addition, the observation that the dimerization domain includes multi-class zinc binding motifs, namely both a zinc finger and a C3HC4 subdomain, has important implications for other C3HC4-containing proteins. The position of this dimerization domain in the N-terminal third of the RAG1 sequence of 1040 amino acid residues may have a significant influence on the activities associated with the C-terminal domains of the protein.

Amino Acid Sequence↗
PubMed1996

Hyperlipidaemia, diet and simvastatin therapy in steroid-resistant nephrotic syndrome of childhood.

In children with steroid-resistant nephrotic syndrome (SRNS) hyperlipidaemia may in the long term be associated with progressive renal insufficiency and increased risk of coronary heart disease. We have assessed the efficacy and tolerability of diet prior to and in combination with a hydroxymethylglutaryl CoA reductase inhibitor, simvastatin, in seven children with SRNS with a mean age of 8 years (range 1.8-16.3 years). Dietary advice to maintain adequate energy and protein intakes with reduced saturated fat and cholesterol intake had little impact on lipid levels pre treatment (mean reduction in cholesterol 1 mmol/l, triglyceride 1.1 mmol/l) but was maintained throughout the study duration. The mean cholesterol and triglyceride concentrations pre treatment were 12.1 +/- 2 (SEM) mmol/l and 8 +/- 2.1 (SEM) mmol/l, respectively. On a median simvastatin dose of 10 mg/day (range 5-40 mg) there was a 41% reduction in cholesterol to 6.6 +/- 0.77 (SEM) mmol/l and a 44% reduction in triglyceride to 3.9 +/- 1.38 (SEM) mmol/l at 6 months which was sustained at 12 months in five patients. The drug was well tolerated with no clinical side effects being noted. Over 6 months the mean plasma albumin concentrations increased from 18.2 +/- 1.26 (SEM) g/l to 23 +/- 2.51 (SEM) g/l, accounted for by three patients (1 complete remission, 1 partial remission, 1 end-stage renal failure). Plasma creatinine concentrations remained stable in five patients with two having progressive chronic renal failure. Growth parameters for both weight and height were maintained. Simvastatin has a beneficial effect on abnormal lipid levels in SRNS but the effectiveness of long-term therapy needs to be evaluated.

Adolescent↗
PubMed1995

Dependence of the phosphorylation of alkaline phosphatase by phosphate monoesters on the pKa of the leaving group.

The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROPO3(2)-(R = 2,4-dinitrophenyl, 4-nitrophenyl, phenyl, glucose-1, glycerol-1, methyl, ethyl, and dodecyl), catalyzed by Escherichia coli alkaline phosphatase and a mutant enzyme, Ser102Cys, have been studied at alkaline pH using the rates of change in the 31P NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (O-Tris phosphate) as the assay. The kcat at pH 8.0 for the wild-type enzyme is approximately 30 s-1 and is independent of the nature of the R group, when the pKa of the leaving group is < 10. Under these conditions the rate of phosphorylation is much faster than dissociation of inorganic phosphate, 15-60 s-1. If the pKa of the leaving group is between 10 and 15, phosphorylation and dissociation of the product phosphate both contribute to the rate limit. If the pKa of the leaving group is > 15, phosphorylation is rate limiting. A Bronsted plot of log kcat vs pKa of the leaving group for those substrates for which phosphorylation is rate limiting yields a beta lg of approximately -0.6. In contrast to the wild-type enzyme, the log kcat values for the S102C mutant enzyme catalyzing the hydrolysis of phosphate esters are linearly dependent on the pKa's of the leaving group throughout the range of pKa from 4 to 16. Phosphorylation of C102 is the rate controlling step, and kcat is independent of the Tris concentration as predicted for rate limiting phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1995

UME6, a negative regulator of meiosis in Saccharomyces cerevisiae, contains a C-terminal Zn2Cys6 binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner.

UME6 is a protein of 836 amino acids from Saccharomyces cerevisiae that acts as a repressor and activator of several early meiotic genes. UME6 contains, near the C-terminus, the amino acid sequence-771C-X2-C-X6-C-X6-C-X2-C-X6-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cys-rich DNA binding subdomains of fungal transcription factors. This sequence has been shown in GAL4 and other proteins to form a zinc binuclear cluster. In spite of the different location, the C-rich sequence, cloned and over-produced within the last 111 amino acid residues of UME6, UME6(111), forms a binuclear cluster and exhibits a Zn-dependent binding to the URS1 DNA sequence. The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6. UME6(111) contains 1.8 +/- 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 +/- 0.1 mol Cd/mol protein. At 5 degrees C, 113Cd2UME6(111) shows two 113Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for 113Cd2GAL4(149). The magnitude of these chemical shifts suggests that each 113Cd nucleus is coordinated to four -S- ligands, compatible with a 113Cd2 cluster structure in which two thiolates from bridging ligands. The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URS1 sequence than the zinc binuclear cluster domain alone. DNase I footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URS1 sequence.

Amino Acid Sequence↗
PubMed1995

Solution structure of the Kluyveromyces lactis LAC9 Cd2 Cys6 DNA-binding domain.

The Zn2Cys6 DNA-binding domain has been identified by sequence homology in approximately forty fungal proteins, including the K. lactis LAC9 transcriptional activator. Using 1H NMR spectroscopy, we have determined the solution structure of a cadmium-substituted form of the LAC9 DNA-binding domain. We have complemented this approach by applying a series of 113Cd-1H NMR experiments, including several novel heteroTOCSY-based techniques. The DNA-binding domain forms a core of two alpha-helix/extended strand segments around the Cd2 binuclear cluster, with a network of amide proton-cysteinyl S gamma hydrogen bonds stabilizing the cluster. Comparison with other Zn2Cys6 domain structures provides insight into the common structural elements used in metal coordination and DNA binding.

Amino Acid Sequence↗
PubMed1994

DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*).

The DNA binding domain of the GAL4 transcription factor from yeast is located in the N-terminal 60 residues of the polypeptide of 881 amino acids. This domain binds 2 Zn ions, which form a binuclear cluster, Zn2C6, with 6 C residues, two of which bridge the 2 metal ions (Gardner KH et al., 1991, Biochemistry 30:11292-11302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recognize the specific DNA sequence, UASG, to which GAL4 binds as a dimer. Gel retardation assays have been utilized to determine the relative affinities of the Zn2 and Zn1 forms of the N-terminal 149 residues of GAL4, GAL4(149*), for UASG DNA sequences. We show that Cd2- and Zn1GAL4(149*) bind to UASG DNA with 2-fold and 4-8-fold lower affinities than Zn2GAL4(149*), respectively. Thus, the metal species and the number of metal ions bound have measurable effects on the specific DNA binding affinity of GAL4, but these differences are small in comparison to the ratio, > 10(3) under some conditions, that characterizes the specific to nonspecific DNA binding affinities of the N-terminal fragments of GAL4. A shorter N-terminal fragment, GAL4(62*), although it continues to recognize the UASG sequence with a high degree of specificity, binds with 1,000-2,000-fold lower affinity than does Zn2GAL4(149*). Gel retardation titrations of a DNA containing 2 UASG sites with increasing concentrations of GAL4(62*) generate a series of 4 retarded bands in contrast to 2 retarded bands formed when the same DNA is titrated with GAL4(149*). These data suggest that GAL4(62*) binds to the UASG sites as individual monomers that dimerize on the DNA, whereas GAL4(149*) binds the UASG DNA cooperatively as a dimer. The approximately 10(3) lower affinity of GAL4(62*) for the UASG DNA can be accounted for by its failure to form dimers in solution. Zn2-, Zn1-, or Cd2GAL4(149*) induces differential rates of gel migration in a series of circularly permutated UASG-containing DNA restriction fragments. Analysis of the data suggests that all 3 proteins cause a 26 degrees angle of bend in the DNA when bound to 1 UASG site and 45 degrees when bound to 2 tandem UASG sites. The same assay shows that GAL4(62*) does not induce significant bending of the UASG DNA sequences. Thus, the additional subdomains found in the larger polypeptide fragment, GAL4(149*), must exert an additional force on the DNA either through direct contacts with the DNA or indirectly through altered protein conformation.

Base Sequence↗
PubMed1994

113Cd-1H heteroTOCSY: a method for determining metal-protein connectivities.

113Cd-1H NMR correlation experiments have been extremely useful for determining the amino acid ligands that form metal-binding sites in proteins. To date, the majority of these methods have used heteronuclear multiple-quantum transfer as the basis for establishing correlations. In this paper, we demonstrate the feasibility of using correlation methods that employ heteronuclear cross-polarization (heteroTOCSY) as viable alternatives. Additionally, we couple heteroTOCSY with selective excitation and transfer procedures to take advantage of the small number of heteronuclei usually present in metalloprotein systems. One- and two-dimensional experiments are presented as examples of these techniques.

Cadmium↗
PubMed1994

Hypertensive adolescents detected by a school surveillance programme: a problem of obesity.

BP has been measured in secondary school children in Nottingham as part of a routine health appraisal since 1988. School nurses who have received appropriate training refer children with a BP greater than the 95th centile for age and sex (Task Force USA 1987) initially to a community paediatrician or general practitioner. Of 14,570 children checked, only 23 patients (14 female) with a mean age of 13.9 years (range 10-15.8 years) were referred to a paediatric nephrology clinic to be seen in consultation with a dietitian. In 12 of 23 children there was a family history of hypertension and in seven a family history of other cardiovascular disease. No patient with secondary hypertension was identified; 14 patients (61%) were overweight or obese (nine) on the basis of percentage weight for height at the time of referral. BP values have tended to normalise on follow-up. Although six of 22 patients lost weight with dietetic support, eight patients gained weight. The school surveillance programme for BP is felt by the school nurses to be a valuable health educational tool and has resulted in few hospital referrals. Although initial dietetic advice may be of value, obesity remains a significant problem for the majority of adolescents in this group.

Adolescent↗
PubMed1992

Conformational changes of HIV reverse transcriptase subunits on formation of the heterodimer: correlation with kcat and Km.

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) is initially expressed as a 66-kDa protein and is subsequently proteolytically processed in vivo to form a 66-kDa/51-kDa heterodimer. Comparison of circular dichroism spectra of the 66-kDa, 51-kDa, and heterodimeric forms of RT indicates that the conversion is accompanied by dramatic changes in subunit conformation. The mean residue ellipticity per subunit at 220 nm decreases from -10.7 x 10(3) deg cm2 dmol-1 for the 66-kDa protein to -6 x 10(3) deg cm2 dmol-1 for the heterodimer. The same loss of ellipticity is observed whether the heterodimer is produced by proteolysis or by mixing a separately-expressed cloned 51-kDa subunit with the 66-kDa protein. Comparison with the spectrum of the cloned 51-kDa protein suggests that much of the conformational change arises from formation of the 51-kDa subunit but substantial changes occur in the remaining 66-kDa subunit as well. A kinetic analysis was performed to correlate these conformational changes with changes in enzyme function. Application of an integrated Michaelis-Menten equation to the catalysis of poly(dT) formation using a d(pT)20-poly(rA) primer-template shows that the kcat for the heterodimer is approximately half that of the 66 kDa enzyme, decreasing from 2.9 to 1.2 nucleotides/s upon formation of the heterodimer. However, km values for the primer-template decrease from 0.54 to 0.12 microM upon heterodimer formation. Thus, kcat/Km is 2-fold larger for the heterodimer, giving it a distinct catalytic advantage at undersaturating concentrations of enzyme and primer-template.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1992

Optical activity associated with the sulfur to metal charge transfer bands of Zn and Cd GAL4.

The N-terminal DNA-binding domain of the GAL4 transcription factor, isolated as a proteolytic fragment of 63 amino acid residues, GAL4(63), or cloned as a 62-residue subdomain, GAL4(62*), binds 2 Cd(II) ions. Both Cd derivatives show intense ellipticity bands associated with -S(-)-->Cd charge transfer bands at 250 nm (+), 233 nm (-), and 218 nm (+). Molar ellipticity values are (5-7) x 10(4) deg.cm2.dmol-1. The energies and pattern (+,-,+) of these ellipticity bands are similar to those observed for the Cd(II) clusters of metallothionein. The latter are believed to be due to the presence of bridging cysteine ligands (Willner, H.W., Vasak, M., & Kägi, J.H.R., 1987, Biochemistry 26, 6287-6292). Circular dichroism provides further evidence that the GAL4 DNA-binding domain forms a binuclear Cd cluster with the amino acid sequence -C11-X2-C14-X6-C21-X6-C28-X2-C31-X6-C38- found in GAL4(62*). Zn2GAL4(62*) has much weaker ellipticity associated with the higher energy -S(-)--> Zn charge transfer bands. Removal of the metal ions from GAL4(62*) results in substantial changes in the near UV circular dichroism due to both loss of the charge transfer bands and changes in the conformation of the peptide backbone. Binding of the UASG enhancer DNA element to either Zn or Cd GAL4(62*) alters the ellipticity of the -S(-)--> metal charge transfer bands suggesting that DNA binding is associated with some changes in conformation around the metal cluster.

Base Sequence↗
PubMed1992

Structure and mechanism of alkaline phosphatase.

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaline Phosphatase↗
PubMed1992

Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins.

In the past five years there has been a great expansion in our knowledge of the role of zinc in the structure and function of proteins. Not only is zinc required for essential catalytic functions in enzymes (more than 300 are known at present), but also it stabilizes and even induces the folding of protein subdomains. The latter functions have been most dramatically illustrated by the discovery of the essential role of zinc in the folding of the DNA-binding domains of eukaryotic transcription factors, including the zinc finger transcription factors, the large family of hormone receptor proteins, and the zinc cluster transcription factors from yeasts. Similar functions are highly probable for the zinc found in the RNA polymerases and the zinc-containing accessory proteins involved in nucleic acid replication. The rapid increase in the number and nature of the proteins in which zinc functions is not unexpected since zinc is the second most abundant trace metal found in eukaryotic organisms, second only to iron. If one subtracts the amount of iron found in hemoglobin, zinc becomes the most abundant trace metal found in the human body.

Amino Acid Sequence↗
PubMed1992

Gastrostomy buttons for feeding children on continuous cycling peritoneal dialysis.

The promotion of growth in infants and young children with chronic renal failure (CRF) requires an aggressive approach to feeding, often in combination with early dialysis. Supplementary feeding has usually involved the use of nasogastric tubes, but these can have many problems. We report our experience with a gastrostomy button device (Bard Ltd.) for long term feeding. Ten children (7 male) had an initial gastrostomy catheter inserted at a median age of 2.0 years (range 0.25-8.5 years). None of the children required an operation for gastrooesophageal reflux and 6 had placement of the gastrostomy catheter at the time of insertion of the Tenckhoff catheter for continuous cycling peritoneal dialysis (CCPD). The catheter is usually exchanged for a similar sized (18 gauge) button device after 4 weeks. All ten children received CCPD in addition to overnight feeding using an enteral feeding pump. The buttons have been in use for a mean of 12 months (range 2-33 months) and are only changed if the anti-reflux valves fail. Nutritional goals have been achieved and growth parameters maintained or improved in 9 children. The button has many advantages over nasogastric tubes or gastrostomy catheters. It has been welcomed by our families in reducing the stress of feeding these young children.

Catheters, Indwelling↗
PubMed1992

Micronutrient supplementation in children on continuous cycling peritoneal dialysis (CCPD).

Data on the micronutrient (vitamin and trace mineral) requirements of children on chronic peritoneal dialysis is limited. Few preparations are of suitable content and palatability. In a prospective study we have assessed and compared the serum levels and dietary intakes of micronutrients (vitamins A, E, B12, folate and zinc, copper, iron) in 7 children on CCPD who were receiving either Ketovite tablets (vitamins C, E and B complex) and Cholecalciferol or a more comprehensive supplement, Paediatric Renal Seravit (vitamins A, E, D, C and B complex with trace minerals). All children received nutritional supplements orally or via a gastrostomy button. These supplements contributed significantly to their nutritional intakes. The mean dietary intakes of the studied micronutrients, with the exception of vitamins A, E, B12 and folate, were below RDA (USA) values. The Renal Seravit was tolerated by only 5 of the 7 patients. There was no significant difference in serum levels of the micronutrients while on the Paediatric Renal Seravit compared to Ketovite. Serum iron levels remained low on both supplements. A comprehensive micronutrient supplement may still be required in children on prolonged dialysis.

Child↗
PubMed1991

Structure of the binuclear metal-binding site in the GAL4 transcription factor.

The GAL4 transcription factor from yeast contains within its N-terminal DNA-binding domain an amino acid sequence containing six cysteine residues, C11-X2-C14-X6-C21-X6-C28-X2-C31-X6-C38. The six Cys residues will form a binuclear metal cluster with either Zn(II) or Cd(II) in which two of the -S- donors are bridging ligands between the two metal ions. Binding of Zn(II) or Cd(II) to the GAL4 DNA-binding domain is essential to induce the conformation of GAL4 required for the protein to recognize the specific DNA sequence, UASG, to which GAL4 binds. Evidence for the presence of the binuclear cluster has come from 113Cd NMR and 2D 1H-113Cd heteronuclear NMR studies of the cloned DNA-binding domain of GAL4 consisting of the N-terminal 62 residues, GAL4(62*) [Pan and Coleman (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Cd(II) binding to the GAL4 DNA is highly cooperative, thus the Cd2Cys6 cluster is always formed. On the other hand, Zn(II) forms well-defined Zn1 and Zn2 complexes with the DNA-binding domain of GAL4, both of which bind specifically to the UASG DNA sequence. The structural details of the Cd2-, Zn2-, and Zn1GAL4(62*) proteins have been determined by a variety of heteronuclear and 2D NMR techniques. When Cd(II) is exchanged for Zn(II), the cluster appears to expand to accommodate the larger Cd(II) ion as suggested by changes of 2 to 4 Hz in the 3JHN alpha coupling constants for the amino acid residues which form the polypeptide loops enclosing the cluster, residues 10-40. These changes suggest alterations in the backbone phi torsional angles of from 20 degrees to 30 degrees. A metal-ligand structure derived from the 1H-113Cd heteronuclear NMR as well as the polypeptide backbone connectivity around the cluster as determined from short-range 1H-1H NOE's is presented. The metal ions also determine the major folding of GAL4(62*), since the chemical shift dispersion in the entire NH-alpha CH fingerprint region of the 1H-1H COSY spectrum collapses on removal of the metal ion. Two short segments of the GAL4(62*) polypeptide (residues 14-19 and 30-36 in the cluster forms, 12-19 and 30-36 in the Zn1 species) show significant dNN(i,i + 1) NOE's. These short segments of polypeptide chain are the only ones that could be helical in the GAL4(62*).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗