Vectorial aspects of 2,3-diphospho-D-glycerate: 2-phosphohydrolase in the human red cell membrane.
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Adenine Nucleotides↗
Biomedical subjects
B D Gomperts
Publications and source records attributed to B D Gomperts.
Demonstration of an enzyme variant in a case of congenital methaemoglobinaemia.
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Blood Protein Electrophoresis↗
Metabolic changes in human red cells during incubation of whole blood in vitro.
1. Whole blood was incubated at 37 degrees , while being dialysed against a large volume of iso-osmotic bicarbonate buffer, pH7.4. The buffer contained glucose and the essential inorganic components of blood plasma in proportion. 2. After 3hr. of incubation in vitro there is a loss of red-cell 2,3-diphosphoglycerate. 3. Isotope experiments show that this is due to an accelerated rate of destruction of this compound. 4. Simultaneously, there is an increase in the median of red-cell osmotic fragility. 5. After extended periods of incubation there is a decrease in the metabolic rate and a decrease in the ratio of the rates of lactate production to glucose consumption. 6. There is a continuous loss of total adenine nucleotide and, after the first 12hr. of incubation, a tendency for the intracellular Na(+) and K(+) to equilibrate with the plasma. 7. The standard deviation of red-cell osmotic fragility expressed among the red-cell population increases exponentially with the time of incubation.
Journal Article↗
Ionomycin stimulates mast cell histamine secretion by forming a lipid-soluble calcium complex.
The activation of cellular function following the direct introduction of Ca2+ into the cytosol by the use of a Ca2+-transporting ionophore has served to confirm the widely held idea that Ca2+ has the status of a second messenger in many cell types. However, this evidence has been obtained largely from the use of a single ionophore, the antibiotic A23187; experiments with X537A, which is another ionophorous antibiotic capable of transporting Ca2+ (ref. 3), have failed to show the expected characteristics. For example, histamine release from rat mast cells mediated by X537A is neither dependent on extracellular Ca2+ nor prevented by metabolic inhibitors. Ionomycin is a recently described polyether antibiotic produced by Streptomyces conglobatus ATCC 31005, and is active against Gram-positive bacteria. The antibiotic action is presumably due to its ionophorous properties, as it extracts Ca2+ from an aqueous phase into an organic phase with a stoichiometry of 1:1 (ref. 6). The ionophore is also capable of transporting 45Ca2+ across biological membranes (our unpublished results). Here we report the application of ionomycin to rat mast cells. We show that ionomycin stimulates mast cell secretion solely through its ability to form a lipid-soluble calcium complex, and thus to convey Ca2+ across the hydrocarbon region of the cell membrane.
Animals↗
Involvement of guanine nucleotide-binding protein in the gating of Ca2+ by receptors.
The introduction of impermeant aqueous solutes into individual cells by microinjection has long been established but the difficulties of manipulating the cytosol composition of large populations of microscopic cells have only recently been overcome. Successful techniques include a dielectric breakdown procedure, treatment with micromolar concentrations of ATP4- (ref. 7) and also with very small (that is nonagglutinating, non-fusogenic) amounts of Sendai virus. So far, attention has been concentrated on the behaviour of the cells (generally their response to applied Ca2+ buffers) at the time when the membrane permeability lesions are open, and thus cytosol and external medium are in contact. I now report a novel technique for monitoring the state of molecular solute permeability in cell membranes and show that the lesions generated by ATP4- in the membrane of mast cells can be closed within seconds of adding Mg2+ so that a cycle of permeabilization and resealing can be used to explore the effect of foreign compounds trapped in the cytosol of effectively intact cells. I show that non-hydrolysable GTP analogues, introduced into the cytosol of mast cells, cause them to undergo exocytotic secretion in response to addition of extracellular Ca2+. This finding is discussed in the light of previous experience relating guanine nucleotide regulatory proteins as intermediaries between receptors and the transducers which they control.
Adenosine Triphosphate↗
Capacitance measurements reveal stepwise fusion events in degranulating mast cells.
Mast cells undergo an extensive and violent morphological transformation on stimulation. Here we describe the dynamics of fusion of the secretory granules in individual mast cells during exocytosis. The cell membrane capacitance (proportional to the cell surface area) was measured using the whole-cell patch-pipette technique, in which the intracellular space is dialysed with the solutions used to fill the patch pipette. Our results show that degranulation occurs spontaneously and reproducibly if the GTP analogue, GTP-gamma-S, and Mg-ATP are present in the pipette filling solution. Contrary to previous reports, in these conditions Ca2+ (and/or Ca2+ buffers) is not required for degranulation. Although electrogenic Ca2+ entry was not detected before or during degranulation and membrane conductance remained low, the capacitance, and by implication the area of the membrane of degranulating cells, increased sigmoidally and stepwise. We conclude that stepwise increases of capacitance are due to the fusion of individual secretory granules with the plasma membrane, and that guanine nucleotide regulatory proteins are involved in the control of this process.
Animals↗
Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase.
Interaction of ligands with 'Ca2+-mobilizing' receptors is thought to result in the generation of two second messengers, inositol trisphosphate and diacylglycerol, from a common substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (refs 1, 2), a component of plasma membranes. It is not known how the occupation of such receptors is translated into the activation of the catalytic unit polyphosphoinositide (PPI) phosphodiesterase, and then to cellular activation, but our recent experiments suggest that GTP regulatory proteins may be involved. In mast cells, non-hydrolysable analogues of GTP introduced and then trapped in the cytosol are able to substitute for external ligands in inducing exocytosis, a well-defined Ca2+-dependent process, suggesting that guanine nucleotide regulatory proteins may act by stimulating the catalytic activity of the PPI phosphodiesterase. We now provide evidence that mast cell secretion is inhibited by internalized neomycin, a compound known to interact with PPI. We also show that the PPI phosphodiesterase of human neutrophil plasma membranes can be activated simply by adding GTP analogues in the presence of concentrations of Ca2+ that pertain in unstimulated cells. These findings strongly support the idea that the coupling factor linking receptor and PPI phosphodiesterase is a guanine nucleotide binding protein analogous to those involved in the activation and inhibition of adenylate cyclase.
Animals↗
Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils.
The term 'stimulus-secretion coupling' has, since first enunciated, been held to involve the mobilization of cytosol Ca2+, which in turn is sufficient to trigger exocytotic secretory processes in metabolically competent cells. However, recent studies on a wide range of secretory cell types indicate that a role for Ca2+ can be obviated: examples are stimulation with phorbol ester (phorbol myristate acetate, PMA) which causes the activation of protein kinase C or the stimulation of platelets with collagen. Ca2+-independent exocytosis also occurs when analogues of GTP are injected through the lumen of patch pipettes directly into the cytosol of mass cells. The results presented here suggest that GTP analogues can activate secretory processes by actions at two distinct locations: one may be at the level of the receptor involving the activation of polyphosphoinositide (PPI) phosphodiesterase with consequent liberation of diacylglycerol (DG); the other involves direct activation of the exocytotic mechanism. These conclusions are based on measurements of exocytotic secretion from permeabilized neutrophils into which we have been able to introduce, individually and in combination, Ca2+ chelators (EGTA and BAPTA), Ca2+ (buffered at micromolar concentrations with EGTA), analogues of GTP and GDP and the direct activator of protein kinase C, PMA.
Animals↗