Subcellular location of glycolytic enzymes inTrypanosoma brucei culture form

Broman, K.; Ropars, M.; Deshusses, J.

doi:10.1007/bf02327033

Cited by 9 publications

(1 citation statement)

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“…Since valinomycin interacts directly with bisoxonol in the presence of K + [11], gramicidin was used to depolarize the cells completely ( Figure 1A). Digitonin (8 µg\ml for procyclics ; 1 µg\ml for bloodstream form) [7,15,16] and nystatin (procyclics, 12-20 µg\ml) [4,17], frequently used to permeabilize selectively the plasma membrane of trypanosomes, also induced an increase in fluorescence characterizing depolarization of the cells (results not shown). Bisoxonol, being negatively charged, is known to be excluded from the mitochondrion and to probe selectively the plasma-membrane potential.…”

Section: Optimization Of the Methodsmentioning

confidence: 96%

Kinetic study of the plasma-membrane potential in procyclic and bloodstream forms of Trypanosoma brucei brucei using the fluorescent probe bisoxonol

Defrise-Quertain

Fraser-L’Hostis

Coral

et al. 1996

Biochemical Journal

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The characteristics of the plasma-membrane potential of procyclic and bloodstream forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent anionic probe bisoxonol. Observation of a stable and representative plasma-membrane potential in the resting state required careful washing, centrifugation and maintenance of the cells at room temperature before measurement. Bloodstream forms were more prone to depolarization during washing at 4 degrees C than procyclic cells. The higher fluorescence observed in the presence of long slender cells than in the presence of procyclic cells shows that the plasma-membrane potential is more negative in the insect form. Healthy dilute cells can sustain their plasma-membrane potential for hours in the presence of external glucose. The presence of a high K+ concentration in the medium did not promote by itself the depolarization of either type of cell. Study of bisoxonol fluorescence as a function of time allowed us to follow the kinetics of the action of metabolic inhibitors in the presence of various ions. o-Vanadate (1 mM) was found to depolarize bloodstream-form cells rapidly but only in a phosphate-free NaCl buffer. Omeprazole and strophanthidin also specifically depolarized bloodstream-form trypanosomes. However, NN'-dicyclohexylcarbodi-imide depolarized both types of cell, but more rapidly for bloodstream-form cells. Bloodstream-form trypanosomes appear to use mainly a vanadate-sensitive Na+ pump to maintain their Na+-diffusion gradient. However, most of the ATPase inhibitors tested had little or no effect on the plasma-membrane potential of procyclics suggesting that this form of trypanosome may rely on several regulation mechanisms.

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