Citrate Efflux in Penicillium simplicissimum: Fundamental Methods for the in vivo Study of Efflux Kinetics
Hubert Firler, Martin Gallmetzer, Wolfgang Burgstaller, Franz Schinner
Institute of Microbiology, Technikerstrasse 25, 6020 Innsbruck, Austria
Received September 25, 1998
Accepted June 17, 1998
Penicillium, citrate efflux, intracellular pH, iniracellular water, disintegration of hyphae, simple diffusion of undissociated citric acid
Several fundamental methods were developed in order to allow a reliable interpretation of in vivo kinetics of citrate efflux in Penicillium simplicissimum. Filamentous growth was achieved by growing P. simplicissimum at pH = 7.3 in a medium containing 1 M HEPES. The volume of intracellular water was determined by thermo-gravimetry as 1.3 mL/g dry wt. A comparison of different methods for extracting citrate from the hyphae showed that boiling the hyphae in 0.1 M HC1 for ten minutes was the most suitable method. Cooling xoas not necessary to maintain the intracellular citrate concentration during harvesting. An optimized method was developed for measuring the cytoplasmic pH. From the calculation of the chemical gradient of undissociated citric acid it was concluded that simple diffusion of undissociated citric acid is of little importance concerning citrate excretion by P. simplicissimum.
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