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Biomedical subjects

R W King

Publications and source records attributed to R W King.

At least 19 recordsLinked to original sources
PubMed1992

19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli.

A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.

Bacterial Proteins↗
PubMed1992

Preparation and properties of an affinity support for purification of cyclic AMP receptor protein from Escherichia coli.

Reaction of cyclic AMP with 1,1'-carbonyldiimidazole produces an intermediate which reacts with primary amines to provide a stable 2'-O-carbamyl derivative. This chemistry has been used to tether cyclic AMP to a Sepharose gel. The resulting affinity support has been used to effect a simple, nondenaturing purification of cyclic AMP receptor protein from crude cell extracts.

Affinity Labels↗
PubMed1991

19F NMR evidence for interactions between the c-AMP binding sites on the c-AMP receptor protein from E. coli.

The 19F NMR spectra of 3-fluorotyrosine containing c-AMP receptor protein (CRP) from E. coli have been recorded in the presence of increasing amounts of c-AMP. One of the signals (from Tyr B) shifts upfield by 0.6 ppm in the presence of excess c-AMP and shows both slow and fast exchange behaviour during the titration. This is evidence for interactions between the two c-AMP binding sites on the CRP dimer leading to different dissociation rate constants (less than or equal to 75 s-1; greater than or equal to 350 s-1) for complexes containing one and two c-AMP molecules.

Binding Sites↗
PubMed1990

19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli.

Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3',5'-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5'-AATGTGAGTTAACTCACATT-3' and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic AMP↗
PubMed1990

Orange/black discolouration of the bowel (at laparotomy) due to clofazimine.

Clofazimine, a commonly used anti-lepromatous drug, is now being prescribed for the treatment of pyoderma gangrenosum, a complication of inflammatory bowel disease. This drug can cause an obstructive exacerbation of Crohn's disease. Surgeons should be aware of the orange/black discolouration of the bowel, which may mimic ischaemia macroscopically. A case, the first reported in Australia, is described and the literature discussed.

Adult↗
PubMed1987

Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli. Optical and ligand-binding studies.

cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6-NH2 group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent. The 2-NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent.

Bacterial Proteins↗
PubMed1986

Alloviroidin, the naturally occurring toxic isomer of the cyclopeptide viroidin.

A novel toxic cyclopeptide from Amanita suballiacea (Murr.) mushrooms that possesses structural features similar to viroidin is described. This peptide, alloviroidin, is identical with viroidin in mass, affinity for actin, and all amino acids except for one. The single discernible difference between the two peptides exists in the configuration at carbon 4 of the 4,5-dihydroxyleucine residues, as shown by a combination of chemical modification and magnetic resonance experiments. The configuration of this residue in viroidin is similar to that of phalloidin and is 2S,4R, while that in alloviroidin is established to be 2S,4S. This peptide is thus unique in its hydroxylation pattern among both the virotoxins and phallotoxins and may be an intermediate for more highly hydroxylated virotoxins, such as viroisin.

Actins↗
PubMed1986

Identification of the 1H resonances of valine and leucine residues in dihydrofolate reductase by using a combination of selective deuteration and two-dimensional correlation spectroscopy.

Lactobacillus casei dihydrofolate reductase (Mr 18 500) contains 16 valine and 14 leucine residues. By comparing the 2D COSY NMR spectra of normal and [gamma-2H6]valine enzyme we have been able to identify all 60 methyl resonances from these residues, and to connect the pairs arising from the same residue. This pairing of the methyl resonances was aided by the examination of the 2D RELAY spectrum which also allowed the C alpha H resonances (and hence the complete spin systems) of 14 of the valine residues to be identified. The combination of selective deuteration with 2D NMR techniques is shown to be a powerful general method for resolving 1H resonances in the complex spectra of proteins and for assigning them to amino-acid type.

Deuterium↗
PubMed1986

Television and video technology in the education of deaf children.

This paper describes a survey into the use of television and related visual media in the education of hearing-impaired children in the UK. The main purpose of the survey was to furnish background information for the development of a low-cost video subtitling system for use in special schools and in units for hearing-impaired children in ordinary schools. An effective method of reducing cost is to base the system on equipment that schools and units already have. The survey indicates that domestic-type video recorders are becoming almost universal. Suitable microcomputers for running subtitling software are also widespread. Other results of the survey reveal: (i) widespread and fairly heavy usage of schools television programmes; (ii) the need for various visual aids and for considerable effort on the part of teachers in making the programmes accessible to hearing-impaired children; (iii) a significant demand for subtitling equipment; (iv) higher incidence of more sophisticated television equipment (video cameras, teletext receivers) and of human technical support in special schools than in hearing-impaired units.

Adolescent↗
PubMed1979

Neoprontosil binding to carbonic anhydrase. Reasonance Raman and other studies on the ionization behavior of the sulfonamide.

Alkalimetric, spectrophotometric, NMR, and resonance Raman titrations are reported for the sulfonamide Neoprontosil in aqueous solution. An assignment of the magnetic resonance peaks for each of the Neoprontosil protons has been made. Neoprontosil is shown to have two "coupled" iity of the microscopic pKs for these two groups precludes spectroscopic characterization of the separate -SO2NH2, -O- or -SO2NH-, -OH species. For this reason, no conclusion can be drawn on the ionization state of the drug when bound to carbonic anhydrase. The resonance Raman spectrum of Neoprontosil bound to human carbonic anhydrase B at pH 9.5 shows a shift in the intense -N=N- stretching mode from 1414 (free) to 1407 cm- (bound), suggesting that a slight conformational change about the -N=N- single bond linkages occurs upon binding.

Carbonic Anhydrases↗
PubMed1979

Acute reversible renal failure secondary to renal candidiasis.

A diabetic patient presenting with bilateral enlarged kidneys and acute renal failure was found to have renal parenchymal candidiasis without obstruction. Treatment with amphotericin B resulted in return of renal function and decrease in renal size.

Acute Kidney Injury↗
PubMed1979

The protective effect of cimetidine on stress-induced acute gastric ulceration in the rat.

The effect of intraperitoneal cimetidine, an H2 receptorantagonist, has been assessed on the development of cold-restraint induced acute gastric ulcers in rats. Cimetidine in doses ranging from 20-100 mg per kg body weight significantly reduced the incidence of acute gastric ulceration compared with saline controls in this model. The protective effect of cimetidine suggests a prophylactic role for this agent in stress-induced gastric or duodenal ulceration in man.

Animals↗
PubMed1978

Urinary zinc excretion following infusions of zinc sulfate, cysteine, histidine, or glycine.

Zinc sulfate (40 mg elemental zinc) or one of three amino acids (5 or 10 g cysteine, 10 g histidine, or 10 g glycine) was infused into anesthetized dogs over a 60-min period. Plasma concentrations and urine excretions of zinc and five other cations, and glomerular filtration rates were determined before, during, and after these infusions. Infusions of zinc sufficient to produce a 20-fold increase in total plasma zinc concentrations had little effect on urinary zinc excretions (threefold increase) or plasma ultrafilterable zinc concentrations. Ten grams of cysteine urinary zinc excretions more than 100-fold, 5 g of cysteine increased zinc excretions more than 30-fold, 10 g of histidine increased zinc excretions sixfold, and 10 g of glycine had no effect. Cysteine infusions appeared to produce a net tubular secretion of zinc. Only histidine appeared to increase serum ultrafilterable zinc concentrations significantly. It would appear that plasma and urine concentrations of certain amino acids, specifically cysteine and histidine, along with polypeptides and other metabolites containing these amino acids, may be major determinants of urinary zinc excretion.

Animals↗