Browsing by Author "Schroder, H. C."
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- A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2Publication . Lorenz, B.; Munkner, J.; Oliveira, M. P.; Leitão, José; Muller, W. E. G.; Schroder, H. C.A method for determining inorganic polyphosphate, which is based on the Mn2+-induced quenching of the fluorescence of the calcium indicator fura-2, is described. The effect of Mn2+ ions on fura-2 fluorescence is gradually abolished in the presence of increasing concentrations of polyphosphate; this allows the quantification both of synthetic polyphosphates and of the naturally occurring polymer isolated from tissues or cells. The described method has some advantages compared to conventional procedures for detection of polyphosphates based on the metachromatic effect on toluidine blue. It can be applied for the determination of pyrophosphate, tripolyphosphate and other short-chain polyphosphates not detectable by toluidine blue and it can be used for measurement both of pyrophosphatase and exopolyphosphatase activity. (C) 1997 Academic Press.
- Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosisPublication . Lorenz, B.; Munkner, J.; Oliveira, M. P.; Kuusksalu, A.; Leitão, J. M.; Muller, W. E. G.; Schroder, H. C.Age-dependent studies show that the amount of inorganic polyphosphate in rat brain strongly increases after birth. Maximal levels were found in 12-months old animals. Thereafter, the concentration of total polyphosphate decreases to about 50%. This decrease in the concentration of total polyphosphate is due to a decrease in the amount of insoluble, long-chain polyphosphates. The amount of soluble, long-chain polyphosphates does not change significantly in the course of ageing. In rat embryos and newborns, mainly soluble polyphosphates could be detected. In rat liver, the age-dependent changes are less pronounced. The changes in polyphosphate level are accompanied by changes in exopolyphosphatase activity, which degrades the polymers to orthophosphate; highest enzyme activities were found when the polyphosphate level was low, Induction of apoptosis in the human leukemic cell line HL-60 by actinomycin D results in degradation of long polyphosphate chains. The total polyphosphate content does not change significantly in apoptotic cells.
- Osmotic-stress-induced synthesis and degradation of inorganic polyphosphates in the alga phaeodactylum- tricornutumPublication . Leitão, J. M.; Lorenz, B.; Bachinski, N.; Wilhelm, C.; Muller, W. E. G.; Schroder, H. C.The potential role of inorganic polyphosphates in the response of algal cells to osmotic stress was investigated. We show that changes in polyphosphate metabolism of algae induced by osmotic stress can be easily determined by measuring the metachromatic shift of toluidine blue produced by polyphosphate and by analysing the size of the extracted polyphosphate on urea/polyacrylamide gels. The amount and size of polyphosphate were found to differ considerably among different algal species. It is demonstrated that the alga Phaeodactylum tricornutum responds to hyperosmotic stress with a marked elongation of polyphosphate molecules and a decrease in the total amount of extractable polyphosphate, while exposure to hypoosmotic stress results in an increase in soluble shorter-chain polyphosphates and a rise in total polyphosphate content.
- Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncuriumPublication . Mangel, A.; Leitão, J. M.; Batel, R.; Zimmermann, H.; Muller, W. E. G.; Schroder, H. C.A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.