The Gram negative bacterium Thauera selenatis is able to respire with selenate as the sole terminal electron acceptor, utilising a periplasmic selenate reductase enzyme to reduce selenate to selenite. Previous characterisation of this enzyme has shown that it is a heterotrimeric molybdo-enzyme (SerABC) of the dimethylsulfoxide reductase family, containing a Mo-bis molybdopterin guanine dinucleotide co-factor, Fe-S clusters and a b-type haem (Schroder et al., 1997, J Biol Chem, 272: 23765-68, Dridge et al., 2007, Biochem J, 408: 19-28). In order to elucidate the electron transport pathway to selenate reductase, and how it can generate a proton motive force, detailed study was required. Firstly, the redox potential of the b-haem of SerC was determined by optical redox titration to be +234 mV. The serC gene was cloned and expressed heterologously in E. coli, but the protein was incorrectly folded into inclusion bodies, and attempts to refold and reconstitute SerC with haem were unsuccessful. A profile of c-type cytochromes in T. selenatis was undertaken, and characterisation of a number of cytochromes was carried out. Two cytochromes were purified, cytc7 and cytc4, and cytc4 was shown to be able to donate electrons to SerABC in vitro. Protein sequence was obtained by N-terminal sequencing and LC-MS/MS, and assigned cytc4 to the cytochrome c4 family of dihaem cytochromes. Redox potentiometry combined with UV-visible and electron paramagnetic spectroscopy showed that cytc4 is a dihaem cytochrome with a redox potential of +282 mV and both haems are predicted to have His-Met ligation. To investigate the role of membrane bound cytochromes in selenate respiration, PCR with degenerate primers amplified a partial gene coding for quinol: cytochrome c oxidoreductase (QCR). A microplate growth method was developed to monitor growth of T. selenatis under reproducible conditions, and used to analyse the effect of respiratory chain inhibitors on growth under different conditions. Aerobic metabolism was unaffected by QCR inhibitors, while nitrite reduction was totally inhibited, linking nitrite reduction to the generation of a proton motive force by the QCR. The QCR inhibitor myxothiazol partially inhibited selenate respiration, showing that some electron flux is via the QCR, but total inhibition of selenate respiration was achieved by combining myxothiazol with the more general inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). These data suggest that electron transfer to selenate reductase occurs via a branched pathway, in which one route is inhibited by myxothiazol and the other by HQNO. Electron transfer via a QCR and a dihaem cytochrome c4 is a novel route for a member of the dimethylsulfoxide reductase family of molybdo-enzymes.

No description available.

A highly sensitive method, based on the use of a microscope fluorimeter and fluorogenic substrates, is being developed to measure rates of drug-metabolising enzyme reactions in small areas of cells in thin liver sections. The results described here illustrate a method in which reaction rates were measured in small groups of approximately 25 hepatocytes. The development of the method has been based initially on the measurement of the cytochrome. P-450-mediated ethoxyresorufin O-deethylase reaction in mouse hepatocytes. Fluorogenic substrates are known, however, for several other drug-metabolising enzymes and alternative fluorogenic substrates, namely pentoxyresorufin and benzyloxyresorufin, for different forms of P-450 have been used in this study. There has been significant improvement over an earlier method (Burke et al., 1983) of sectioning non-fixed, non-frozen pieces of liver. The left lobe of a mouse liver was stuck with cyanoacrylate glue to the Teflon stage of the Vibraslice cutting machine. The Teflon stage and attached liver lobe were immersed in a bath of Krebs-Henseleit physiological buffer. The specimen bath was moved towards the vibrating blade which cut through the liver so producing a section which was transferred with a fine brush to the surface of a slide of encapsulating resin on to which it was pinned. The section of liver was then superfused with the Krebs-Henseleit solution at 37 C on the heated stage of a microscope. The tip (10-12 um diameter) of a micropipette containing a solution of ethoxyresoruf in DMSO was manoeuvred onto the surface of the section using a micromanipulator. Selection of the cells to be measured and placement of the micropipette were carried out in transmitted light. The microscope was then switched to epi-illumination fluorescence mode. Ethoxyresoruf in was expelled from the micropipette on to the section by the application of a 2-5 second pulse of pressurised Nitrogen gas (approximately 10 lbf/in2). The volume of ethoxyresoruf in expelled was estimated as 0.405 0.027 ml using a combination of [3Hj-harmine and 1 mM ethoxyresorufin in the micropipette. The spread of substrate and the ensuing O-dealkylation reaction were confined to a circular area of the tissue approximately 100 um in diameter, comprising about 25 hepatocytes. During, the reaction, the fluorescence of the forming metabolite, resorufin, was measured continuously by means of a computerised photomultiplier system attached to the microscope. The measurement of fluorescence was from the surface layer of cells only. Since the method is at a stage in development, various, pieces of apparatus were subject to change. The excitation and emission filters were chosen so that the selected wavelengths of light were more appropriate for the metabolite resorufin than the substrate ethoxyresorufin. For excitation, a combination of a Vickers OG515 long wave pass filter (50% transmission between 370 nm and 560 nm), a Vickers TRITC exciter (50% transmission between 380 nm and 560 nm) and a Balzer TRITC dichroic mirror (10% transmission between 490 nm and 570 nm) were used. For emission, a Balzer TRITC dichroic mirror and a Barr and Stroud DL7S narrow band pass filter (maximum transmission of 49.2% at 590.1 nm) were used. The filters provided epi-illumination (incident) excitation light of 515-560 nm and selected emitted fluorescence of 580-600 run. A photomultiplier converted the photons of light into an electrical signal which was converted to a digital signal. Ideally the photomultiplier tube in use at each of the improvements was maximally sensitive to the emission wavelengths of resorufin. The digital signal was displayed either on a pen recorder or on a microcomputer, both of which resulted in a graph of relative fluorescence against time. From this graph three measurements were taken. These were: i) the initial reaction velocity (rate of linear rise of fluorescence intensity). ii) the highest fluorescence intensity attained, and iii) the time to reach the highest fluorescence intensity after the cessation of ethoxyresorufin application. The initial velocity had a closer correlation with the maximum reading (r = 0.75) than with the time at which the maximum occurred (r = 0.43). The results encompassed a wide range of values but did follow a normal distribution which meant that the Student's t-test could be used to test for significant differences between means. Means s.e. mean initial velocities measured in centrilobular and periportal regions of liver sections from control mice, were 2.7 0.32 and 1.9 0.28 fluor. unitsmin-1 respectively, suggesting no' difference between centrilobular and periportal regions. Pretreatment with MC increased these values to 55.0 + 3.5 and 49.9 3.3 respectively but again suggested no centrilobular/periportal difference, -Naphthoflavone produced less of an effect causing an increase from 11.2 1.7 to 52.9 10.6 in centrilobular regions and from 7.3 1.0 to 51.7 10.0 in periportal regions.

No description available.

The metabolism of drugs and foreign compounds is a major influence on their pharmacological and toxicological effects. The haemoprotein cyt. P-450 exists as a multi-isozyme family which functions in the metabolism of a wide variety of foreign compounds. Biochemical information concerning the structure and catalytic function of cyt. P-450 isozymes from man is less extensive than from animal species. In this study several microsomal proteins including four cyt. P-450 isozymes, NADPH-cyt. P-450 reductase, epoxide hydrolase and other unidentified proteins were purified from adult human liver; the methodologies previously employed to isolate cyt. P-450 isozymes were improved and now allow better resolution and recovery of human cyt. P-450. Comparison of the physical and chromatographic properties of the cyt. P-450 isozymes isolated (cyt. P-450 7:3 cyt. P-4507:4 and cyt. P-4507:5) indicated that they were very similar and probably identical. N-terminal amino acid sequence and immunochemical data demonstrated that these isozymes probably belong to the steroid-inducible gene family. Further characterisation of cyt. P-4507:3 indicated that it is a major inducible isozyme in man and that it metabolises the calcium entry blocker nifedipine but not a structurally similar drug nicardipine.

1. The molecular weights of the G6PD subunits from the three yeast sources, bovine adrenals and rat liver were estimated by SDS-PAGE and found to be in the region of 55-59 K. An estimate of 46 K was similarly made for the G6PD subunit from the prokaryote, L. mesenteroides. 2. Two close bands or sometimes one diffuse band appeared on the polyacrylamide slab gel following electrophoresis and Coomassie blue staining for rat liver G6PD. These corresponded to the molecular weight region of about 57-59 K. So far, the evidence from peptide sequences does not reveal more than one subunit sequence. 3. G6PD from bakers' yeast was inactivated with sodium [1-. C] acetylsalicylic acid, following whichit was shown that 1.1 mole of 14 C moiety had been incorporated per enzyme subunit and that a lysine residue, essential to enzyme activity, had been modified. 4. Inactivation of bakers' yeast G6PD in the presence of high concentrations of substrate or coenzyme indicated that the acetylsalicylic acid was binding to the enzyme at a site which was directly or indirectly involved in substrate binding. The observation that high concentrations of coenzyme in the incubation mixture did not offer protection against acetylsalicylic acid inhibition was supplemented by TRNOE studies which showed no significant change in the conformation of coenzyme bound to bakers' yeast G6PD inactivated with acetylsalicylic acid compared with the active enzyme. 5. Acetylsalicylic acid did not inactivate rat liver G6PD to the same extent as the bakers' yeast enzyme. Inactivation of rat liver 14 G6PD with C-HIAB was relatively slow and resulted in the partial labelling of many cysteine residues. 6. Sequence studies on rat liver G6PD resulted in the isolation of a peptide which was homologous in sequence to the bakers' yeast G6PD tryptic peptide containing the reactive lysine residue, although no evidence for special reactivity of the corresponding lysine residue in the rat liver enzyme was found. 7. A high degree of sequence homology was noted between rat liver G6PD and human erythrocyte G6PD. It was also established that the human erythrocyte G6PD sequence published by Beutler (1983) was incorrect due to the misalignment of tryptic peptides.

Ph.D. (Botany) / Please refer to full text to view abstract

Low molecular weight gelators (LMWGs) have gained much attention over the last few decades, because of their ability to form supramolecular architectures as well as their many potential applications in biomedical research and as advanced materials. Most of the gelators were discovered through serendipity, and their structural requirements are somewhat ambiguous. This is due, in part, to the fact that the supramolecular gelation phenomenon is not yet fully understood, though many structural classes have been found to be excellent organogelators. Carbohydrates are abundant natural resources that are useful in preparing advanced materials. We have previously showed that monosaccharide derivatives can form effective low molecular weight gelators for both organic solvents and aqueous mixtures. In this research, we have studied the gelation capability of several glucosamine derivatives. Several series of 4,6-O-acetal protected glucosamine derivatives were synthesized and screened for their gelation properties in several solvents.

The HIV/AIDS still remains a global health challenge with 42 million people infected with the virus. An alarming 70% of these people reside in sub-Saharan Africa with HIV-1 subtype C being the most prevalent subtype in this region and globally. HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of the HI virus. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug resistant strains. It has been postulated that disruption/dissociation of the dimer interface may lead to an inactive enzyme. The development of small molecules and peptides has been a major research area with the key target being the N- and C-termini antiparallel β-sheet. This is due to its highly conserved nature and because it consists of a cluster of amino acids that contribute most of the binding energy and stability of the dimer interface. Hence it is referred to as a ‘hot-spot’. Therefore, binding of protease inhibitors at this site could cause destabilisation and/or dissociation of the enzyme. The terminal residue, F99, was mutated to an alanine disrupting the presumed lock-and-key motif it forms and in turn creating a cavity at the N- and C-termini antiparallel β-sheet. A second mutant, W42F/F99A, was created for monitoring tertiary structural changes exclusively at the N- and C-termini antiparallel β-sheet. The F99A and W42F/F99A, compared to the wild-type, showed a higher expression yield and also migrated further when separated using tricine SDS-PAGE. Wild-type protease CD spectra showed a minimum at 214 nm and a local maximum at 230 nm, while the mutants exhibited minima at 203 nm and absence of the local maxima. A 50% higher fluorescence intensity and a 2 nm red-shift for the mutants versus the wild-type was observed. According to SE-HPLC data the relative molecular weight of the wild-type, F99A and W42F/F99A are 16.4 kDa, 20.7 kDa and 18.1 kDa, respectively. Although the thermal unfolding of all three proteases was irreversible, the unfolding transition of the wild-type was clearly defined between 55 °C and 63 °C. The F99A and W42F/F99A unfolding curves were linear without clearly defined transition states. The specific activity of the F99A (0.13 μmol/min/mg) amounted to a ten-fold reduction compared to the wild-type (1.5 μmol/min/mg). The substrate binding affinity (KM) for the F99A was 41% lower than the wild-type when 2 μM of protein was used. The Vmax and kcat values were about 30-fold and two-fold, respectively, higher for the wild-type when compared to the F99A. Therefore, the tricine SDS-PAGE analysis, secondary and tertiary structural characterisation and thermal denaturation curve showed that the F99A mutation has altered the structure causing ‘partial’ unfolding of the protein. But, the protein still maintained minute activity. The overlap between the ANS binding spectra of the wild-type and variants suggests that the dimeric form still exists.

The Human Immunodeficiency Virus (HIV) subtype C accounts for the majority of infections in Southern Africa. The HIV protease is one of the targets in HIV treatment due to its pivotal role in HIV maturation in the host cell. However, because of polymorphisms in the HIV genome, drug resistance becomes a major problem in HIV treatment. Polymorphisms in the HIV protease gene result in altered substrate cavities, and /or flap hinge modifications leading to unfavourable drug interaction with the enzyme. The most common form of drug resistant mutations is single amino acid substitutions. Although, amino acid insertions have been reported, this form of mutation in the HIV protease is rare. L38↑N↑L insertion is a unique form of HIV protease polymorphism that was isolated from a patient failing drug therapy in South Africa. The objective of this research was to assess the impact of the L38↑N↑L insertions, with accompanying background mutations, on the structure and function of this form of polymorphism in HIV-1 South African subtype C protease. The far-UV circular dichroism (CD) spectra of L38↑N↑L protease shows a trough at 203 nm, suggesting alterations in the secondary structure content of this mutant. Whereas the wild type (WTCSA-HIVPR) displays a trough at 215 nm. However, tertiary structure characterisation using fluorescence spectroscopy did not detect changes within the local tryptophan environment of L38↑N↑L protease in comparison with the wild type due to no significant shift in emission wavelength. The specific activity of L38↑N↑L protease and wild type was 28.0±1.3 μmol.min-1.mg-1 and 123.45±6.4 μmol.min-1.mg-1 respectively. The turn-over number for L38↑N↑L protease and wild type was 1.0 × 10-3 ± 6.0 × 10-5 and 7.7 × 10-3 ± 5.6 × 10-4 respectively. As much as the presence of known drug resistance mutations in L38↑N↑L can be attributed to drug resistance, it should also be noted that the insertions may have also caused local structural alterations that may have enhance drug resistance of L38↑N↑L. These changes could have lead to the decreased catalytic activity of the L38↑N↑L protease. Homology modelling studies show that the insertions in L38↑N↑L protease may have resulted in a fold similar to 2HS1 (PDB code), which has a modification on the flap hinge. In addition, the homology modelling studies suggest that L38↑N↑L protease may have a second inhibitor binding site next to one of the flap hinge regions as seen in the 2HS1 model. In conclusion, the L38↑N↑L insertions and accompanying background mutations may have contributed to the local structural modifications that lead to drug resistance in L38↑N↑L protease.