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Biomedical subjects

M J Crabbe

Publications and source records attributed to M J Crabbe.

At least 19 recordsLinked to original sources
PubMed1991

Aldose reductase inhibitors and cataract.

Blindness in diabetics is largely due to retinopathy and/or cataract. Hyperglycaemia and the duration of diabetes are major risk factors for the development of cataract and retinopathy. This review details some of the reactions of glucose that are relevant to the development of complications, and follows the elucidation of monosaccharide autoxidation and its relevance to the aldose reductase reaction and its determination. Inhibitors of this 'aldose reductase' reaction are shown to have a number of effects which may be of importance to their action in vivo. The pharmacological implications of chemotherapy for diabetics with complications are briefly discussed.

Aldehyde Reductase↗
PubMed1991

Nonlinear steady-state kinetics of chloramphenicol acetyltransferase.

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.

Acetyl Coenzyme A↗
PubMed1991

Aldehyde dehydrogenase, aldose reductase, and free radical scavengers in cataract.

Human lens was found to contain aldehyde dehydrogenase at a level of activity similar to that of bovine lens, namely 1.76 +/- 0.51 IU/g. The enzyme, which appears to be a tetramer of 229 kD, was less susceptible to inhibition by cataractogenic agents than the bovine enzyme. The lipid peroxidation product malondialdehyde was a good substrate of the human lens enzyme. The in vitro aldose reductase reaction, which we have shown is caused by glyceraldehyde-stimulated free-radical NADPH oxidation, is inhibited by the potential anti-cataract agents, bendazac acid and bendazac lysine; these compounds also inhibit ferricytochrome c reduction in the presence of DL-glyceraldehyde and scavenge superoxide radicals. These results are consistent with the hypotheses that aldehyde dehydrogenase is a protective enzyme in the human lens, and that the peroxy radical scavenging effects of bendazac acid and bendazac lysine contribute to their anti-cataract activity.

Adult↗
PubMed1990

Modelling of poliovirus. HIV-1 antigen chimaeras.

We have used laboratory-based molecular modelling to identify structural features of antigen chimaeras of poliovirus expressing epitopes from human immunodeficiency virus (HIV-1) that may affect virus viability. Chimaeras were constructed by replacement of antigenic site 1 of VP1 by sequences corresponding to epitopes from HIV-1. Loop volume, estimated by approximating the loop to an ellipsoid was significantly (P less than 0.001) lower in viable (2062.1 A3 +/- 400.2) than in non-viable (3617 A3 +/- 650.7) constructs. Our results suggest that viable virus will only be formed when antigen chimeras modified at antigenic site of VP1 have a loop occupying a similar volume in space to that occupied by the antigenic site 1 loop. In addition, the modified loop must fit with the peptide bond angles and distances at the top of the beta-barrel of VP1.

Amino Acid Sequence↗
PubMed1988

Simple graphical methods for use with complex ligand-binding and enzyme mechanisms.

Simple graphical plots in distribution-free space are shown to provide estimates of mechanism and kinetic parameters for complex steady-state processes. The methods were tested using simulated 1:1, 1:2, 2:2, 2:3 and 3:3 functions, and real data from polyol dehydrogenase, NADPH oxidation in the presence of DL-glyceraldehyde, and NADPH oxidation in the presence of an NADPH-binding protein. Estimates of mechanism and parameters agreed closely with those found from non-linear regression by computer.

Aldehyde Reductase↗
PubMed1987

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.

Acremonium↗
PubMed1987

A spectrophotometric assay for deacetoxycephalosporin C synthase.

A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for alpha-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.

Acremonium↗
PubMed1987

Kinetic effects of metal ion chelating reagents and their analogues on bovine lens aldehyde dehydrogenase.

The chelating agents 1,10-phenanthroline and 2,2'-dipyridyl gave partial inhibition of the bovine lens aldehyde dehydrogenase, which was fully reversible and competitive towards acetaldehyde. The non-chelating compounds, 1,7-phenanthroline, 4,7-phenanthroline and 4,4'-dipyridyl gave total competitive inhibition, and significant activation at saturating substrate and coenzyme concentrations, in a manner similar to cyanide. All five isomers prevented the binding of the coenzyme NAD+, the non-chelating phenanthroline compounds being the most effective. An overall mechanism for inhibition by single and bidentate ligands is proposed.

2,2'-Dipyridyl↗
PubMed1987

Purification and characterization of cloned isopenicillin N synthetase.

Isopenicillin N synthetase (IPS) cloned from Cephalosporium acremonium has been isolated from transformed Escherichia coli and purified to homogeneity. The resulting, abundant, recombinant protein, whilst undergoing slightly different N-terminal processing to that observed for the fungally-derived protein, has identical kinetics for the conversion of LLD-aminoadipoyl-cysteinyl-valine to isopenicillin N. Recombinant IPS converts analogue substrates into unusual beta-lactam antibiotics in exactly the same way as the fungal protein.

Amino Acid Sequence↗
PubMed1986

Bovine lens aldehyde dehydrogenase: activity and non-linear steady-state kinetics.

Bovine lens aldehyde dehydrogenase is located predominantly in the cortical and nuclear regions, although the specific activity is highest in the epithelial cells. A novel two-step procedure has been used to purify aldehyde dehydrogenase from bovine lens to homogeneity. A comparison using published assay methods for aldehyde dehydrogenases showed that the dimeric lens enzyme had the highest specific activity of any cytoplasmic aldehyde dehydrogenase, although the kcat value was not exceptional. Computer curve-fitting showed that the minimum degree of the rate equation with propionaldehyde and acetaldehyde as substrates was 2:2. The relationship (a2 X b1)/(a1 X b2) was used to show the marked effect of temperature, and to a lesser extent pH, on the non-linear steady-state kinetics. These results indicate that the rate-determining step at low aldehyde concentrations (probably aldehyde binding) is accelerated by increasing temperature to a much greater degree than the rate-determining step at high aldehyde concentration (probably NADH release).

Acetaldehyde↗
PubMed1985

Low apparent aldose reductase activity produced by monosaccharide autoxidation.

Low apparent aldose reductase activity, as measured by NADPH oxidation, can be produced by the spontaneous autoxidation of monosaccharides. NADPH is oxidized to metabolically active NADP+ in a solution of autoxidizing DL-glyceraldehyde at rates of up to 15 X 10(-4) A340/min. The close parallelism between the effects of buffer salt type and concentration, monosaccharide structure and temperature activation on autoxidation and NADPH oxidation imply that autoxidation is a prerequisite for the NADPH oxidation, probably via the hydroperoxy radical. Nucleotide-binding proteins enhanced NADPH oxidation induced by DL-glyceraldehyde, up to 10.6-fold with glucose-6-phosphate dehydrogenase. Glutathione reductase-catalysed NADPH oxidation in the presence of autoxidizing monosaccharide showed many characteristics of the aldose reductase reaction. Aldose reductase inhibitors acted as antioxidants in inhibiting this NADPH oxidation. These results indicate that low apparent aldose reductase activities may be due to artifacts of monosaccharide autoxidation, and could provide an explanation for the non-linear steady-state kinetics observed with DL-glyceraldehyde and aldose reductase.

Aldehyde Reductase↗
PubMed1985

Partial sequence homology of human myc oncogene protein to beta and gamma crystallins.

The human cellular myc gene is one of about 20 cellular oncogenes which code for a variety of proteins including protein kinases and growth factors. The human gene is related to the avian myelocytomatosis leukaemia virus MC29 and produces a binding protein which may be involved in regulation of gene expression and cellular differentiation and proliferation. The crystallins are proteins in the eye lens synthesised at different stages of cell differentiation and proliferation, and whose short range order is necessary for lens transparency. Computer-based sequence comparisons show that beta Bp and gamma II crystallins, which show partial sequence homology and conservation of 'Greek Key' motives are also partially homologous to two regions on the human myc protein, though this protein probably does not conserve the 'Greek Key' structural motives.

Amino Acid Sequence↗
PubMed1985

Partial sequence homologies between cytoskeletal proteins, c-myc, Rous sarcoma virus and adenovirus proteins, transducin, and beta- and gamma-crystallins.

Computer based sequence comparisons indicate partial sequence homology between human c-myc, Rous sarcoma virus, adenovirus 7, and simian sarcoma virus proteins and the cytoskeletal proteins d9smin, keratin and vimentin. In addition, sections of the oncogene proteins showed partial but significant homology to alpha and gamma subunits of transducin. gamma-II and beta-BP crystallins showed partial but significant homology to the cytoskeletal proteins keratin, vimentin, desmin, alpha and beta-tubulin, and to adenovirus 7 and simian sarcoma virus transforming gene proteins. Beta-BP crystallin showed partial but significant homology to Rous sarcoma virus protein, and to alpha and gamma subunits of transducin. Both crystallins showed partial sequence homology to the GTP-binding protein elongation factor TU from Escherichia coli. These sequence homologies suggest a link between the mechanisms of normal lens cell differentiation, involving modifications to the cytoskeleton and subsequent changes to the pattern of protein synthesis, and mechanisms of neoplastic transformation. Furthermore, the transducin-like region on beta-crystallin may be important for its interaction with lens membranes and the maintenance of short-range order for lens transparency.

Adenoviridae↗