Influence of cadmium on lipid peroxidation and antioxidant enzymes in isolated rat hepatocytes

Cadmium (Cd) influences lipid peroxidation (LPO) by enhancing peroxidation of membrane lipids and by disturbing the antioxidant system of cells. In isolated rat hepatocytes, LPO was observed in cells incubated with Cd (50-250 mu M) for various time periods up to 90 min. The antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were inhibited along with depletion of glutathione (GSH) in hepatocytes treated with Cd. The results show that Cd influences LPO in rat hepatocytes due to decrease in antioxidant status.

Protection against UVB inactivation (in vitro) of rat lens enzymes by natural antioxidants

Oxidative damage, through increased production of free radicals, is believed to be involved in UV-induced cataractogenesis (eye lens opacification). The possibility of UVB radiation causing damage to important lenticular enzymes was assessed by irradiating 3 months old rat lenses (in RPMI-1640 medium) at 300 nm (100 mu Wcm(-2)) for 24 h, in the absence and presence of ascorbic acid, alpha-tocopherol acetate and beta-carotene. UVB irradiation resulted in decreased activities of hexokinase, glucose-6-phosphate dehydrogenase, aldose reductase, and Na, K- ATPase by 42, 40, 44 and 57% respectively. While endopeptidase activity (229%) and lipid peroxidation (156%) were increased, isocitrate dehydrogenase activity was not altered on irradiation. In the presence of externally added ascorbic acid, tocopherol and beta-carotene (separately) to the medium, the changes in enzyme activities (except endopeptidase) and increased lipid peroxidation, due to UVB exposure, were prevented. These results suggest that UVB radiation exerts oxidative damage on lens enzymes and antioxidants were protective against this damage.

Synergistic effect of UVB radiation and age on HMPS enzymes in rat lens homogenate

The behaviour of rat lenticular enzymes, glucose-6-phosphate dehydrogena.se (G6PD, EC: 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGD, EC: 1.1.1.44) as a function of age and UVB irradiation (in vitro) was investigated by irradiating the lens homogenate from 3-and 12-month-old rats at 300 nm (100 μW cm 2). In the 3-month-old group the specific activities of G6PD and 6PGD were reduced by 26% and 42%, respectively, after 24 h of irradiation, whereas in the 12-month-old group the decrease was 38% and 49% respectively, which suggests that the susceptibility of HMPS enzymes to UVB damage is higher in older lenses. The decrease in specitic activity was associated with a change in apparent Km and Vmax (marginal in 3 months and significant in 12 months) of these enzymes due to UVB irradiation. UVB irradiation also decreased the levels of NADPH and NADPH/NADP ratio. These changes, altered activities of G6PD and 6PGD and altered levels of NADPH. may in turn have a bearing on lens transparency.

Mechanistic insights into type III restriction enzymes

Type III restriction-modification (R-M) enzymes need to interact with two separate unmethylated DNA sequences in indirectly repeated, head-to-head orientations for efficient cleavage to occur at a defined location next to only one of the two sites. However, cleavage of sites that are not in head-to-head orientation have been observed to occur under certain reaction conditions in vitro. ATP hydrolysis is required for the long-distance communication between the sites prior to cleavage. Type III R-M enzymes comprise two subunits, Res and Mod that form a homodimeric Mod(2) and a heterotetrameric Res(2)Mod(2) complex. The Mod subunit in M-2 or R2M2 complex recognizes and methylates DNA while the Res subunit in R2M2 complex is responsible for ATP hydrolysis, DNA translocation and cleavage. A vast majority of biochemical studies on Type III R-M enzymes have been undertaken using two closely related enzymes, EcoP1I and EcoP15I. Divergent opinions about how the long-distance interaction between the recognition sites exist and at least three mechanistic models based on 1D- diffusion and/or 3D-DNA looping have been proposed.

A novel ``pro-sensitizer'' based sensing of enzymes using Tb(III) luminescence in a hydrogel matrix

Chemically synthesized ``pro-sensitizers'' release the sensitizer in the presence of lipase or beta-glucosidase, triggering a significant luminescence response from a lanthanide based hydrogel.

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

FOLD TYPE II PYRIDOXAL 5 `-PHOSPHATE DEPENDENT ENZYMES: STRUCTURE, SUBSTRATE RECOGNITION AND CATALYSIS

Enzymes utilizing pyridoxal 5'-phosphate dependent mechanism for catalysis are observed in all cellular forms of living organisms. PLP-dependent enzymes catalyze a wide variety of reactions involving amino acid substrates and their analogs. Structurally, these ubiquitous enzymes have been classified into four major fold types. We have carried out investigations on the structure and function of fold type I enzymes serine hydroxymethyl transferase and acetylornithine amino transferase, fold type n enzymes catabolic threonine deaminase, D-serine deaminase, D-cysteine desulfhydrase and diaminopropionate ammonia lyase. This review summarizes the major findings of investigations on fold type II enzymes in the context of similar studies on other PLP-dependent enzymes. Fold type II enzymes participate in pathways of both degradation and synthesis of amino acids. Polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity are briefly described. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes; Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA, The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a. property similar to the eukaryotic type I topoisomerases, The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.

Nonlinear Enzyme Kinetics Can Lead to High Metabolic Flux Control Coefficients: Implications for the Evolution of Dominance

In a classic study, Kacser & Burns (1981, Genetics 97, 639-666) demonstrated that given certain plausible assumptions, the flux in a metabolic pathway was more or less indifferent to the activity of any of the enzymes in the pathway taken singly. It was inferred from this that the observed dominance of most wild-type alleles with respect to loss-of-function mutations did not require an adaptive, meaning selectionist, explanation. Cornish-Bowden (1987, J. theor. Biol. 125, 333-338) showed that the Kacser-Burns inference was not valid when substrate concentrations were large relative to the relevant Michaelis constants. We find that in a randomly constructed functional pathway, even when substrate levels are small, one can expect high values of control coefficients for metabolic flux in the presence of significant nonlinearities as exemplified by enzymes with Hill coefficients ranging from two to six, or by the existence of oscillatory loops. Under these conditions the flux can be quite sensitive to changes in enzyme activity as might be caused by inactivating one of the two alleles in a diploid. Therefore, the phenomenon of dominance cannot be a trivial ''default'' consequence of physiology but must be intimately linked to the manner in which metabolic networks have been moulded by natural selection.