The effect of temperature on bacterial degradation of EDTA in pH-auxostat

The effect of temperature on the maximum specific growth rate and the cell yield was studied during cultivation of two bacterial strains (LPM-4 and Pseudomonas sp. LPM-410) on EDTA under unlimited cell growth conditions in a pH-auxostat. Both strains displayed linear dependence of reciprocal biomass yield against reciprocal specific growth rate, from which the values of rate of substrate expenditure for cell maintenance and the “maximum” yield (i.e., hypothetical yield without cell maintenance processes) were estimated. Analysis of the maximum yield values based on mass–energy balance theory suggested that oxidation of the carboxylic acid side chains of EDTA by a monooxygenase had zero or low energetic efficiency. An Arrhenius equation with different values of Arrhenius parameters within different temperature ranges gave a good fit with the temperature dependence of both growth rate and biomass yield. Specific growth rates of both strains showed a more pronounced temperature dependence than did the cell yields. A possible kinetic mechanism was suggested which might be responsible for the modes of the temperature dependences of specific growth rate and yield that were found. The mechanism is based on a hypothetical key substance governing the metabolic flows, which is formed in a zero-order reaction and destroyed in a first-order reaction, both rate constants depending on temperature according to the Arrhenius law.     

The cellular dynamics of hepatic receptors for alpha 2-macroglobulin.protease complex and for insulin are different

Making freshly isolated rat hepatocytes permeable by 0.4 g/liter digitonin doubled the number of binding sites for alpha 2-macroglobulin.trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surface-exposed in its absence. Upon preincubation of the cells at 37 degrees C, the total cellular binding capacity for alpha 2-macroglobulin.trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37 degrees C, reached after 20 min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for alpha 2-macroglobulin.protease complexes is regulated independently of other specialized plasma membrane proteins.     

2-Deoxy-D-galactose metabolism in ascites hepatoma cells results in phosphate trapping and glycolysis inhibition.

The metabolism of 2-deoxy-D-galactose has been studied in AS-30D rat ascites hepatoma cells in suspension. Using 2-deoxy-D-(1-14C)galactose and an alkaline ethanol deproteinization procedure, the quantitatively identified metabolites included 2-deoxy-D-galactose 1-phosphate comprising 99.3%, and UDP-2-deoxy-D-galactose and UDP-2-deoxy-D-glucose, together amounting to 0.4% of the total metabolites. After incubation for 5 h in the presence of 2-deoxy-D-galactose (1 mmo1/1), the content of 2-deoxy-D-galactose 1-phosphate reached 35 mmo1x(kg cells)-1. The rate of phosphorylation of 2-deoxy-D-galactose was rapid during the first 30 min and decreased to approximately 20% of this rate during the subsequent hours. The rapid trapping of Pi in the form of 2-deoxy-D-galactose 1-phosphate resulted in a depression of free intracellular Pi in spite of a concomitant increase in net 32Pi uptake from the medium and a decrease of ATP and other 5'-nucleotides. The rates of glucose utilization and lactate production were depressed by more than 80% in the presence of 2-deoxy-D-galactose (1 mmo1/1). Interruption of Pi trapping by removal of 2-deoxy-D-galactose from the medium reversed the depressions of Pi and ATP and resulted in a rapid but incomplete relief of glycolysis inhibition. Crossover analysis of glycolytic intermediates indicated an inhibition at the 6-phosphofructokinase step. The depression of glucose utilization may be mediated by the increased level of glucose 6-phosphate, a potent inhibitor of hexokinase. An additional inhibitory effect of a metabolite of 2-deoxy-D-galactose at the 6-phosphofructokinase step was indicated by crossover analysis after reversal of Pi and ATP depressions in the presence of a high intracellular content of 2-deoxy-D-glactose 1-phosphate. The quantitative analysis of the metabolites of 2-deoxy-D-galactose demonstrated the predominance of the monophosphate and the negligible formation of UPD derivatives of this sugar analog in AS-30D hepatoma cells. This provides a system for the investigation of a galactose analog as a phosphate-trapping agent in the virtual absence of uridylate trapping.