Date of Award
Master of Science (MS)
Dr. Charles E. Barr
This study attempts to determine if a fixed stoichiometry of the K+/H+ exchange is present in Nitella clavata. Nitella was cultured with constant aeration under illumination of approximately 2000 lux measured at the culture solution surface, with sixteen hours of illumination alternated with eight hours of darkness. Internodal cells were harvested by excision, with harvested cells ranging from 3-5 cm in length and 700-900 μm in diameter. Isolated cells were then placed in a 5.70 pH K solution and kept in an incubator at 22°C under 350 lux illumination. Cells were preconditioned for seven days before experimentation. H+ extrusion was measured with a Leeds and Northrup pH meter using a small volume pH electrode. Experiments ranged from .75-8 hours. Prior to the measurement, cells were pre-loaded with H+ by placing them in a large volume of K solution at pH 4.7, usually for a period of 85 minutes. Cells were then rinsed in fresh K solution at pH 5.7 for three minutes, followed by a second rinse in fresh K solution for seventeen minutes. After this rinse period, the net H+ efflux in K solution was found to be negligible. Then the cells were placed in a 20x K solution. Net H+ extrusion was measured by addition of 5mM KOH at intervals to keep pH at 5.7. This continued until the rate of pH change of the external K solution became negligible. K+ influxes were measured using the same system with the exception that the 20x K solution was 20x 42K solution. Cl- influxes were measured using the same procedure, with the exception that the 20x K solution contained 36Cl-. Membrane potentials were measured using an Ag+/AgCl glass microelectrode consisting of a chloride silver wire inserted into a 1 mm microcapillary glass tube. The researcher observes that the present study does not indicate the presence of a strict relationship between H+ extrusion to K + influx. Nitella may require a critical amount of H+ accumulation within the cell before any significant quantity of that H+ will become available for net extrusion into the external environment. The 1:1 stoichiometry observed in some cells and tissues does not appear to be present in Nitella clavata. Further work must be done to determine if the observed phenomenon is indeed characteristic of Nitella clavata.
Holland, Daniel John, "H+ and K+ Transport in Nitella" (1980). Biology Master’s Theses. 67.