Showing papers by "Sathyanarayana N. Gummadi published in 2008"

Inducible nature of the enzymes involved in catabolism of caffeine and related methylxanthines.

Abstract: Previously isolated strain of Pseudomonas sp. has the capability of utilizing caffeine as the sole source of carbon and nitrogen and degrading caffeine at higher concentrations (>10 g l–1). In this study, an assay has been developed to study the enzymatic conversion of caffeine to subsequent methylxanthines by cell free extracts of Pseudomonas sp., the activity of which has been stabilized by use of stabilizers in the lysis buffer. Growth of the strain in various methylxanthines and later enzyme assay demonstrated that the enzyme(s) involved in degradation of caffeine and other methylxanthines were inducible in nature. The results also indicated that more than one enzyme are involved in degradation of caffeine to xanthine, which constitute the primary steps in bacterial caffeine catabolism. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants.

Abstract: Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

Over-expression of recombinant human phospholipid scramblase 1 in E. coli and its purification from inclusion bodies.

Abstract: Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (~30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue “Stains-all” dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli.

Growth of Halotolerant Food Spoiling Yeast Debaryomyces nepalensis NCYC 3413 Under the Influence of pH and Salt

Abstract: Debaryomyces nepalensis, a halotolerant food-spoiling yeast could grow in complex (YEPD) medium at different pHs ranging between 3.0 and 11.0 in the absence of salt and at pH 3.0–9.0 in the presence of different concentrations of NaCl and KCl. The specific growth rate of D. nepalensis was not affected by the initial pH of the medium in the absence of salts, whereas it was affected in the presence of salts. At 2 M NaCl and KCl, the organism exhibited a synergistic effect on pH and salt stress, which was unique in the Debaryomyces species. Irrespective of the initial pH and salt, the intracellular pH of D. nepalensis was ~7.0. Significant organic acid was produced at neutral and alkaline pH and organic acid production increased with the increase in pH and salt. Very specific organic acids are produced in the presence of NaCl and KCl. Our observation would contribute to a better understanding of the physiological phenomenon of halotolerance in D. nepalensis.

Chemotaxis of Pseudomonas sp. to caffeine and related methylxanthines.

Abstract: Pseudomonas sp. isolated from soil of coffee plantation area has been shown to degrade higher concentrations of caffeine (∼15 g l–1) by N-demethylation at a rate higher than what has been reported for any strain so far. This strain exhibits positive chemotaxis towards caffeine (1,3,7-trimethylxanthine) in swarm plate assay and modified capillary assay in a dose dependant manner. Related methylxanthines and xanthine also act as chemoattractants for the strain with the highest relative chemotactic response (RCR) seen for xanthine. Chemotaxis in Pseudomonas sp. is possibly plasmid mediated as indicated by positive chemotaxis of plasmid transformed E. coli DH5α. The chemotactic abilities of Pseudomonas sp. combined with higher rates of degradation of caffeine can be used in the development of strategies for biodecaffeination of caffeine containing wastes. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)