ISSN:
1615-6102
Keywords:
Intracellular pH
;
Light/dark
;
Nitella
;
Redox potentiometry
;
Redox state
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Summary The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (ERED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, ERED values of+237±93mVand+419±51 mV with respect to a normal H+-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state ERED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of ERED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic ERED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic ERED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10μM 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), ERED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01360734
