Distortions effects were studied as a function of gel environment, apparatus design and buffer type. Outward lanes distortions pronounced in low conductivity buffers for both continuous buffer systems and stacking gels, were field strength dependent. In discontinuous buffer systems, the moving boundary of the Laemmli buffer system deformed depending on the environment. In the Michov buffer system, the high conductivity resolving gel surrounded by a low conductivity electrode buffer, displayed straight pathways until a field strength of 15 V/cm, permitting to obtain information about lanes distortions depending on the acrylamide matrix structure. A decreased ammonium persulfate concentration, e.g. to 0.03%, induced phase segregation in gels at low temperature during the run: a critical endpoint near 11 degrees C for a gel in the Michov buffer system under conditions of electrophoresis was supposed. Gel collapse resulted in inward deviations in the lanes of the pattern and also faint bands in the zones where the gel was retracted. Besides this effect of catalyst concentration, the inwardly distorted pattern depended on temperature, field strength, solvent and hydrolysis due to gel aging. Amendments for overcoming lane distortions are suggested.

No abstract

No abstract

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.7 mM) (site I), the second one with a low affinity for pyruvate (Kt = 6.1 mM) and a high affinity for sodium (Kt = 23.9 mM) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25 mM and 8 mM pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (delta psi congruent to 0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation was n = 1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives a n = 1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).

No abstract