Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.     

Effects of electron donors on Ca2+-dependent K+ transport in one-step inside-out vesicles from the human erythrocyte membrane

The interactions between reducing agents and Ca2+ in the activation of Ca2+-dependent K+ transport have been studied in one-step inside-out vesicles. The artificial electron donor system ascorbate + phenazine methosulphate increases the apparent sensitivity to Ca2+ by about 5-times over control values (half activation constant, about 5 X 10(-8) M) while oxidized cytochrome c decreases the sensitivity to about 1/3 of the controls. Using redox buffers at a fixed pCa it is shown that the shift from the low to the high-affinity state can be accounted by the reduction of a membrane component accepting two electrons and with an apparent standard redox potential (pH 7.5) of 47 mV. The electrons can be transferred directly from reduced PMS or to oxidized cytochrome c, but not from ascorbate, NADH or reduced glutathione.     

The use of lectins for determination of absolute configurations of small amounts of sugars eluted from chromatograms

A simple procedure for the determination of the absolute configuration (i.e., assignment to the D- or L-enantiomeric series) of glucose, mannose, galactose, fucose, arabinose, and rhamnose is described, based on inhibition by these sugars of 125I-labeled lectin binding to the glycoconjugates immobilized on the wells of plastic microculture plates. The method works well with 10 to 100-micrograms amounts of the sugars isolated after paper chromatography of the glycoprotein or polysaccharide hydrolysates.     

Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation

Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye. EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC). Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation. This finding suggests that their histidines, i.e. His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor.     

The use of a cyanine dye in measuring membrane potential in yeast

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.     

Antilipolytic activity in vitro of various analogs of nicotinic acid. Structure-activity relationship

The antilipolytic activity of a series of N aryl-nicotinamides and of alpha picolinic acid, has been tested in vitro. Lipolysis was stimulated by epinephrine (20 micrograms/ml of incubation medium) using rat's epididymal adipose tissue slices. Only N(2-carboxy methyl phenyl) nicotinamide showed antilipolytic effect comparable to that of nicotinic acid at similar concentrations (2 X 10(-5) M). Picolinic acid (10(-4) M) showed no antilipolytic effect. These results, together with those of the literature, are discussed in regard to the relations between structure and antilipolytic activity.     

Incorporation of (U-14C)-glucose into glycogen in normal rat pancreatic islets

The incorporation of glucose into glycogen was determined in pancreatic islets isolated from normal rats and incubated with glucose (5 or 20 mM) and compounds known to affect glycogen metabolism in other tissues. Incubation of pancreatic islets with glucose (20 mM) induced a marked increase in radioactive glycogen. Exposure to epinephrine in the presence of glucose (20 mM) slightly increased incorporation of glucose into glycogen. In contrast the incorporation of glucose into glycogen was not affected when isolated islets were exposed to glucagon or insulin, whereas anti-insulin serum in the incubation medium decreased radioactive glycogen formation.     

Possible effect of bromocriptine on the insulin receptor

The administration of 0.00011 mg/g weight/day of bromocriptine (CB154) for 7 days to Wistar rats, improved the peripheral glucose uptake without significant changes in plasma insulin level, during the intravenous glucose tolerance test (0.33 g/kg). The mode of the bromocriptine action on binding of 125I insulin to erythrocyte insulin receptors has been evaluated. The total number of sites was greater with bromocriptine (513.1 +/- 124.1 pM/1,CB154 815.6 +/- 107.9 pM/l) (p less than 0.01). The high affinity/low capacity compound of insulin receptor, in CB154 rats (51.8 +/- 16.8 pM/l) was higher than in normal rats (18.3 +/- 8.9 pM/l) (p less than 0.005). Additional studies indicated that CB154 had no effect on the rate of association and dissociation of 125I insulin from rats erythrocyte insulin receptors. The degradation of insulin or the erythrocyte receptor sites do not change, after the treatment with CB154.     

Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii

In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii. Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology. EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions. Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells.