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Biomedical subjects

M C Tejedor

Publications and source records attributed to M C Tejedor.

30 records · Page 2Linked to original sources
PubMed1992

Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity.

PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.

Cells, Cultured↗
PubMed1991

Solubility behaviour of phosphofructokinase from haemolysate of erythrocytes, reticulocytes and bone marrow cells in poly (ethylene glycol) solutions.

The precipitation profiles of phospofructokinase obtained by addition of poly(ethylene glycol) to haemolysates of erythrocytes, reticulocytes and bone marrow are displaced towards higher polymer concentrations when the pH decreases from 6 to 5 or increases from 6 to 8. In the pH range 5 to 8, the concentration of polymer required to provide any level of precipitation follows the order erythrocytic less than reticulocytic less than bone marrow phosphofructokinase. The precipitation of erythrocytic and reticulocytic phosphofructokinase is enhanced by the presence of F6P and ATP. No effect is observed for bone marrow phosphofructokinase. These results are consistent with an isoenzymatic variation of phosphofructokinase in erythrocytes, reticulocytes and bone marrow cells.

Adenosine Triphosphate↗
PubMed1991

Differential solubility behaviour in poly(ethylene glycol) solutions of glucose 6-phosphate and 6-phosphogluconate dehydrogenases from bone marrow, reticulocytes and erythrocytes.

Precipitation of glucose 6-phosphate dehydrogenase by poly(ethylene glycol) depends on both pH and the source of haemolysate. An increase in pH from 5 to 7 leads to an increase in the polymer concentration required for precipitation. At any pH, polymer concentration needed to precipitate the enzyme increases in the order bone marrow less than reticulocytes less than erythrocytes. This differential behaviour seems to be due to variations on the effect of pH on the state of aggregation and/or differences in the intrinsic solubility of the enzyme present in the three haemolysates. In contrast, precipitation profile of 6-phosphogluconate dehydrogenase only slightly moves towards higher concentrations of PEG when raising the pH from 5 to 7, being similar at each single pH in the three haemolysates.

Animals↗
PubMed1990

Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems.

Glucose 6-phosphate dehydrogenase shows a high partition coefficient in poly-(ethylene glycol)-dextran aqueous two-phase systems in comparison with those for 6-phosphogluconate dehydrogenase, phosphofructokinase and the bulk of proteins present in rat erythrocyte haemolysates. As a consequence, fractions highly enriched in glucose 6-phosphate dehydrogenase can be obtained after multiple partitions in the above systems with a counter-current distribution procedure. Phosphofructokinase shows a high affinity for Cibacron Blue and, as a result, the enzyme can be extracted in the top phase of poly(ethylene glycol)-dextran systems containing Cibacron Blue-poly(ethylene glycol) (affinity systems). The efficiency for the purification of the enzymes by partitioning is increased up to 10-fold when enzyme-rich fractions, obtained by precipitation with poly(ethylene glycol), are used instead of original haemolysate. The recovery of enzyme activities is near 100% in both instances.

Animals↗
PubMed1988

Solubility behavior of enzymes after addition of polyethylene glycol to erythrocyte hemolysates.

The addition of polyethylene glycol to a hemolysate of rat erythrocytes reduces the solubility of phosphofructokinase and glucose-6-phosphate and 6-phosphogluconate dehydrogenases in an exponential manner with respect to polymer concentration. Analyses of the solubility curves (log solubility versus polymer concentration) obtained at different pH values suggest that the solubility can be related to both the aggregation state and the intrinsic solubility of the proteins promoted by solution conditions. These findings suggest the possibility of using polyethylene glycol in a rational way for the fractional precipitation of a mixture.

Animals↗
PubMed1986

Selective precipitation of phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from rat erythrocyte hemolysates by polyethylene glycol.

Precipitation profiles of phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been established in the range of 0-16% PEG at different pH values. Precipitation generally occurred between narrow limits of polyethylene glycol. The polymer concentration needed to reach any level of enzyme precipitation is dependent on pH. Particular conditions (% PEG and pH) for the selective enzyme enrichment have been determined.

Animals↗
PubMed1984

Kinetic behaviour and regulatory properties of phosphofructokinase in rat bone marrow cells.

Phosphofructokinase from bone marrow cells shows sigmoidal kinetics with respect to fructose-6-phosphate when studied at near physiological concentrations of ATP (1.5 mM) and pH (7.1). The enzyme is clearly inhibited by ATP concentrations higher than 0.75 mM. pH increases maximum velocity and affinity of the enzyme towards fructose-6-phosphate and decreases the cooperative behavior of the enzyme. Citrate behaves as a negative allosteric effector. ATP deinhibition and activation of bone marrow phosphofructokinase, by either AMP or cAMP, were also observed. cAMP seems to have a higher affinity for the enzyme than AMP. cGMP does not show any antagonistic effect versus cAMP as has been previously observed in rat erythrocytes or reticulocytes.

Adenosine Triphosphate↗