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Transketolase, the most critical enzyme of the non-oxidative branch of the pentose phosphate pathway, has been reported as a novel target in Plasmodium falciparum as it has least homology with the human host. Homology model of P. falciparum transketolase (PfTk) was constructed using the crystal structure of S. cervisiae transketolase as a template, and used for the identification and prioritization of potential compounds targeted against Plasmodium falciparum transketolase. The docking studies with fructose-6-phosphate and thiamine pyrophosphate showed that His31, Asp473, Ser388, Arg361 and His465 formed hydrogen bonds with fructose-6-phosphate while pyrimidine ring of coenzyme interacted with conserved residues of protein viz., Leu121, Glu415, Gly119. The major interacting residues involved in binding of oxythiamine pyrophosphate were similar to cofactor binding site of PfTk. An integrated pharmacophore, co-factor ThDP and substrate fructose-6-pho- sphate, based virtual screening of a small mo- lecule database retrieved eight and thirteen compounds respectively. When screened for their activity against P. falciparum transketolase, one compound in case of ThDP and three compounds in case of fructose-6-phosphate based screening were found active against PfTk. Identification of these novel and chemically diverse inhibitors provides initial leads for optimization of more potent and efficacious drug candidates to treat malarial infection.

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.

Pro-oxidant and anti-oxidant systems and their levels have significant roles in occlusive vascular diseases. In the present communication, we have measured the levels of some representative anti-oxidant enzymes in the blood of the patients of myocardial infarction after reperfusion and compared them to age and sex matched healthy persons. Our findings show that the activities of anti-oxidant enzymes (viz. SOD, catalase and glutathione reductase) are significantly decreased whereas there is significant increase in the levels of malonaldialdehyde (a marker of free radical-mediated damage) in the patients. The findings point out that ischemic myocardial disorders are associated with excessive free radical generation and free radical-mediated damage of lipids.

Plasma membrane (PM) vesicles isolated from the yeast Saccharomyces cerevisiae (wild-type NCIM 3078, and a MG 21290 mutant pma 1-1) were used to monitor the effect of the detergents, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (Chaps) and Triton X-100, on (H+)-ATPase (E.C. 3.6.1.35), NADH oxidase and NADH-hexacynoferrate (III)[HCF (III)] oxidoreductase (E.C. 1.6.99.3) activities. The results obtained show that Triton X-100 inhibited both membrane bound and solubilized NADH-dependent redox activities. The nature of this inhibition as determined for NADH-HCF(III) oxidoreductase was non-competitive and the Ki values for wild and mutant enzymes were 1.2 x 10(-5) M and 8.0 x 10(-6) M, respectively. The findings are interpreted, in view of the established reports, that the active site architecture of PM bound NADH-dependent oxidoreductase in yeast is likely to be different than in other eukaryotes.

The activity of plasma membrane bound redox enzyme, NADH-HCF(III) oxidoreductase, in wild and mutant strains of the yeast Saccharomyces cerevisiae is modulated by Con A in a dose-dependent manner. The solubilized activity is enhanced at lower concentration and inhibited at higher concentration of Con A. The enzyme in mutant strain is more sensitive to inhibition. The activation of enzyme by Con A is suppressed in the presence of either alpha-methyl-D-mannoside or 2-deoxy-D-glucose, indicating the glycoproteic nature of enzyme as well as the resulting conformational change due to interaction with Con A as the factor for modulated activities. This was supported by recording the decrease in K(m) value of enzyme with respect to substrate NADH in the presence of lower concentration of Con A. The purified enzyme was more sensitive to lectin stimulation and, on the basis of comparative stimulatory effects of Con A and PSA on activity, it is likely that mannosyl moiety in enzyme is involved in binding the lectins to cause enzymic activation.

Summary Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca 2+ -uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca 2+ uptake by the protoplasts both in dark and under blue light. The Ca 2+ -calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca 2+ -uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca 2+ -calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta

Summary Redox activities were compared in plasma membrane (PM) vesicles isolated by aqueous polymer twophase partitioning from the roots of chick pea seedlings (Cicer arietinum L. cv. Radhey) grown in hydroponic nutrient medium under deficiencies of boron or zinc. Seedlings raised under mineral-sufficient conditions served as control. The membrane preparations were highly purified and largely in right-side-out orientation as seen by marker enzyme assays, electron microscopy and latency studies of the PM marker, vanadate-sensitive K+-stimulated Mg2+-ATPase, with non-ionic detergents Triton X-100 and Brij 35. PM vesicles from boron and zinc deficient tissues showed lower ATPase and NAD(P)H-dependent redox activities vis-a-vis sufficient-tissue except under zinc deficiency where NADPH-dependent redox activity was higher. Quantitative inhibition of redox activities was seen by calmodulin antagonists (calmidazolium and W-7) in the presence of Triton X-100. The findings indicate that boron and zinc are essential for optimal function of PM ATPase and redox system(s) in chick pea.

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305 microM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.

Platelets play important role in precipitating ischaemic myocardial syndromes in many ways. One of the consequences of ischaemic diseases is excessive generation of oxygen derived free radicals that have numerous pathophysiological consequences. Platelet pro-oxidant enzyme, xanthine oxidase is one of the sources of generation of free radicals. In the present paper, we report the effect of administration of vitamin E along with aspirin on the levels of platelet xanthine oxidase and extent of free radical mediated damage in the patients reperfused after myocardial infarction. Our findings show that administration of 400 mg. vitamin E for six days along with 80 mg. aspirin has an excellent anti-oxidant effect as evidenced by reduced platelet xanthine oxidase activity and lowering of malondialdehdye (MDA) levels which is an index of the extent of free radical mediated damage.

A differential activity peak of pectate lyase (PEL) was observed during ripening of banana fruits (Musa acuminata Harichhal) receiving different hormone treatments. Exposure of fruits to 25 ppm ethylene for 24 h, as well as dipping of M. acuminata fruits in 1 mM 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 h, hastened fruit ripening. Both PEL activity peak and climacteric peak were observed on the 4th and 10th days of treatment with ethylene and 2,4-D, respectively, compared to the 16th day in control fruits. Gibberellic acid (GA) treatment retarded fruit ripening and both PEL activity and climacteric peaks were observed on the 19th day. Treatment of fruits with ethylene or 2,4-D also advanced the appearance of a polygalacturonase (PG) peak and GA delayed its appearance, but the activity peaks always appeared in post-climacteric fruits, in contrast to PEL activity peaks coinciding with the respiratory peaks.

Summary Menadione increased the rate of electron transfer from NADH to O 2 /ferricyanide [HCF(III)] in the presence of plasma membrane (PM) vesicles. This rate was further enhanced by Triton X-100. The latency of NADH oxidase/NADH-HCF(III) reductase indicated that electron donor and acceptor sites are also present on the external face of PM vesicles.

The presence of transplasma membrane electron transport in a variety of plant cells and tissues is reported. It is now agreed that this property of eukaryotic cells is of ubiquitous nature. Studies with highly purified plasma membranes have established the presence of electron transport enzymes. Two types of activities have been identified. One, termed "Standard" reductase, is of general occurrence. The other, inducible under iron deficiency and relatively more active, is "Turbo" reductase. However, the true nature of components participating in electron transport and their organization in the plasma membrane is not known. The electron transport is associated with proton release and uses intracellular NAD(P)H as substrate. The electron flow leads to changes in intracellular redox status, pH, and metabolic energy. The responsiveness of this system to growth hormones is also observed. These findings suggest a role for electron flow across the plasma membrane in cell growth and regulation of ion transport. Involvement of this system in many other cellular functions is also argued.

Summary Trans-plasma membrane electron transport activity, monitored by reduction of exogenous ferricyanide, in cut apical segments of Cuscuta showed a concomitant proton release into the medium. The exposed tissue surface also possessed NADH oxidase activity. Plant growth regulators, viz. GA, N6-BA, 2,4-D, IBA and Etherel had markedly varying effects on the rates of these plasma membrane-linked activities.

Summary Angiospermic parasites viz., Cuscuta reflexa, Dendrophthoe falcata and Orobanche cernua , exhibited a reducing capacity for exogenous ferricyanide in the dark at the exposed plasma membrane surface. This extracellular reduction was not attributed to the extrusion of reductants from tissue segments. There also occurred no uptake of ferricyanide from the incubation medium. Increase in the exposed surface area of the tissue to ferricyanide as well as the exposure to fluorescent light showed a concomitant increase in ferricyanide reduction. Metabolic and ATPase inhibitors, and uncouplers manifested a differential response with the apical segment of Cuscuta in its ability to reduce extracellular ferricyanide. It is suggested that a transplasma membrane electron transfer executed by a membrane-bound oxidoreductase was responsible for the reduction of extracellular ferricyanide.

Ferricyanide is reduced in the dark by whole cells of Anacystis nidulans. A transmembrane redox system spanning the plasma membrane of these cells is proposed to be the mechanism involved in external ferricyanide reduction by these cells. Ferricyanide reduction is not due to the excretion of reductants, as indicated by a low rate of ferricyanide reduction in the supernatant after removal of cells. Low rates of cytochrome c reduction compared to ferricyanide indicate ferricyanide reduction is not due to release of low potential reducing agents. Catechol release by these cells is minimal. The ferricyanide is not reduced after entry into cells, since ferricyanide is recovered in the suspension media. Lack of inhibition by sulfhydryl reagents indicates that the excretion of sulfhydryl reductants or the presence of sulfhydryl sites on the membrane are not causing external ferricyanide reduction. No ferricyanide oxidation by whole cells of A. nidulans was detected. Inhibition of O2-uptake by ferricyanide suggests that a plasma membrane respiratory system may be partially involved. Stimulation of the rate of ferricyanide reduction by 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HOQNO) is a further indication of the selective involvement of the plasma membrane respiratory chain in the external ferricyanide reduction by A. nidulans.

Summary Extracellular reduction of ferricyanide was exhibited by isolated Cuscuta protoplasts. A larger decrease in NADH than NADPH levels of the ferricyanide-treated protoplasts pointed to the major involvement of the former as an electron donor. Glutathione levels were also found to be lowered in similarly treated tissue. The time-dependent variation in intracellular ATP levels in presence of ferricyanide supported the concept of plasma membrane ATPase activation during transplasma membrane electron transport in eukaryotes.

The effects of endosulfan on Rhodotorula gracilis cells are varied and concentration-dependent. They are exhibited as increased rate of respiration, retardation in pH-recovering activity of cell suspensions and loss in the rate or D-xylose uptake. The temperature dependence of sugar uptake indicated that endosulfan reacts with some membrane component(s).

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K+ (86Rb+) transport into cells, but it did not affect sucrose transport. N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K+ (86Rb+) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K+ (86Rb+). p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K+ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K+ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K+.

Cells of the yeast Rhodotorula glutinis on treatment with N,N′-dicyclohexylcarbodiimide (DCCD) at a concentration of about 0.5 mM fail to accumulate d -xylose, cause efflux of accumulated sugar and do not exhibit H+/sugar symport. The results are interpreted as being due to depolarization of the membrane potential by DCCD.