Biomedical subjects

J L Slonczewski

Publications and source records attributed to J L Slonczewski.

21 records · Page 2Linked to original sources

PubMed1985

Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1982

Effects of pH and repellent tactic stimuli on protein methylation levels in Escherichia coli.

Intracellular pH (pH(int)) and extracellular pH (pH(ext)) of Escherichia coli were measured at 12-s time resolution by (31)P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH(ext) (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH(int) decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s(-1) cm(-2) pH unit(-1). Addition of the membrane-permeant weak acid benzoate at constant pH(ext) also caused a lowering of pH(int); at high concentrations it generated an inverted transmembrane pH gradient (DeltapH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH(int) was lowered by weak acid addition or when pH(ext) was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH(ext) 7.0). The weak-acid concentration needed to reach that limit was dependent on pH(ext), as would be expected on the basis of DeltapH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH(int), demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH(int) and pH(ext). Evidence is presented that pH(int) may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-(3)H- and (35)S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.

Acetates↗

PubMed1981

pH homeostasis in Escherichia coli: measurement by 31P nuclear magnetic resonance of methylphosphonate and phosphate.

The intracellular pH of Escherichia coli cells, respiring on endogenous energy sources, was monitored continuously by 31P NMR over an extracellular pH range between 5.5 and 9. pH homeostasis was found to be good over the entire range, with the data conforming to the simple relationship intracellular pH = 7.6 + 0.1(external pH - 7.6) so that the extreme values observed for intracellular pH were 7.4 and 7.8 at external pH 5.5 and 9, respectively. As well as inorganic phosphate, we employed the pH-sensitive NMR probe methylphosphonate, which was taken up by glycerol-grown cells and was nontoxic; its pKa of 7.65 made it an ideal probe for measurement of cytoplasmic pH and alkaline external pH.

Cell Membrane↗