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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.