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PubMed2004

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase.

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.

Binding Sites↗
PubMed1982

The steady-state transport of cationized ferritin by endothelial cell vesicles.

1. The steady-state transfer of cationized ferritin by endothelial cell vesicles has been investigated quantitatively using electron microscopy. Single capillaries from the mesenteries of decerebrated frogs were perfused in vivo with solutions containing 3-5 g 100 ml(-1) cationized ferritin or cationized ferritin (3-5 g 100 ml(-1)) and bovine albumin (1 g 100 ml(-1)). Perfusions lasted between 60 and 240 s, at which time the tissues were fixed in situ with osmium tetroxide.2. Measurements of the free diffusion co-efficient of cationized ferritin in the presence and absence of 1 g 100 ml(-1) albumin (0.400+/-0.09 x 10(-6) cm(2) s(-1) and 0.361+/-0.08 x 10(-6) cm(2) s(-1), respectively) were not significantly different which suggests that albumin does not bind to cationized ferritin. Together they yielded a value for the Stokes-Einstein radius of cationized ferritin of 5.59 nm, which was not significantly different from that of native ferritin.3. Examination of transverse sections of perfused capillaries showed a layer of cationized ferritin molecules (> 26 nm thick) close to the luminal surface of the endothelial cell wall, in both the presence and absence of albumin. Estimates of the concentration of cationized ferritin within the layer showed it to be approximately twice that of the perfusate concentration, confirming that cationized ferritin binds and concentrates at the cell surface.4. When no albumin was present in the cationized ferritin perfusate, all the luminal vesicles (those open to the luminal cell surface) were labelled with molecules of cationized ferritin. The mean number of ferritin molecules per labelled luminal vesicle (F/N(L)) was 5.0+/-0.7, a value close to that predicted from the concentration of cationized ferritin in the layer if it was assumed that the whole of the vesicle volume was available to molecules of cationized ferritin, i.e. that cationized ferritin could penetrate the cell coat lining the vesicles as it does that covering the luminal cell surface.5. Few cytoplasmic vesicles (0.26+/-0.04) and abluminal vesicles (0.07+/-0.03) were labelled with cationized ferritin in the absence of albumin. The mean number of cationized ferritin molecules per labelled vesicle in both vesicle populations was also low. In addition, labelled cytoplasmic vesicles F/N(L) = 1.87+/-0.33) always contained significantly fewer ferritin molecules than labelled luminal vesicles (F/N(L) = 5.0+/-0.7). These findings offer further support for the fusion model of the steady-state transfer of ferritin by endothelial cell vesicles (Clough & Michel, 1981) and are not consistent with the translocation of labelled luminal vesicles across the cell. They also suggest that cationized ferritin binds to the cell coat lining the vesicles, and is unavailable for transfer during transient fusions between vesicles.6. The presence of albumin in the cationized ferritin perfusate reduced the fractional labelling of all three vesicle populations to one third of their values in its absence. It also reduced the mean number of ferritin molecules per labelled vesicle at all three sites in the cell. It is suggested that albumin reduces the volume of distribution of cationized ferritin within the vesicles either by competing with cationized ferritin for the same binding sites within the cell coat, or by simply occupying space within the extracellular matrix.

Animals↗
PubMed1990

Modulation of dopamine receptors by cations in 7315a, MtTW15 and estradiol-induced pituitary tumors.

Modulation of dopamine (DA) receptors by cations (Na+, K+, Mg2+, Ca2+) was compared in 7315a, MtTW15, and estradiol valerate-induced (EV-T) pituitary tumors, and intact adenohypophysis. In 7315a tumors, the affinity of [3H]spiperone binding measured at 25 degrees remained unchanged in the presence of each cation individually or all these cations together (IONS) compared to the affinity obtained using a buffer without ions; the density (Bmax) was not affected by monovalent cations or Mg2+ and was decreased by Ca2+ or IONS. When binding experiments were done at 37 degrees, monovalent cations increased affinity whereas divalent cations or IONS did not modify it, and none of these cations affected Bmax values. In MtTW15 tumors, the affinity of [3H]spiperone binding measured at 25 degrees was not changed by Na+ or IONS and was decreased by K+ or divalent cations; the density was decreased by K+ and unchanged by all the other cations. When binding experiments were done at 37 degrees, Na+ increased the affinity, whereas all the other cations did not affect it: the density was unaffected by all the cations studied. In EV-T assayed at 37 degrees, the affinity was increased by monovalent cations or Mg2+ and was unchanged by Ca2+; monovalent cations did not affect the density of [3H]spiperone binding and divalent cations increased it. In binding experiments performed at 25 degrees and 37 degrees, choline chloride did not change the affinity or the density of [3H]spiperone binding to DA receptors in the three pituitary tumors investigated, suggesting that the effect of cations was specific and not due to differences in ionic strength. In the rat normal anterior pituitary, Na+ increased the affinity of [3H]spiperone for the DA receptors, whereas the affinity was unchanged by Ca2+; the density of [3H]spiperone binding was unaffected by these cations. Our results suggest that DA receptors in 7315a and MtTW15 tumors are regulated abnormally by sodium, potassium, magnesium and calcium. In contrast, DA receptors in EV-T are regulated normally by monovalent cations and abnormally by divalent cations as compared to these receptors in intact pituitary tissue.

Animals↗
PubMed1995

A non-selective cation current activated via the multifunctional Ca(2+)-calmodulin-dependent protein kinase in human epithelial cells.

1. Activation of macroscopic membrane currents by intracellular calcium ([Ca2+]i) signalling pathways was examined in human T84 epithelial cells, a model secretory cell line. 2. Elevation of [Ca2+]i by either the calcium ionophore A23187 (1 microM) or the cholinergic agonist carbachol, led to the transient activation of both a chloride and cation current in single voltage clamped cells. The channels underlying the cation conductance were found to be equally permeable to external Na+, K+ and Cs+, but impermeable to the large organic cations tetraethylammonium and N-methyl-D-glucamine (NMDG). These observations indicate that the cation channels are non-selective with respect to monovalent cations. 3. Persistent activation of both the chloride and non-selective cation currents by [Ca2+]i was observed following inhibition of cellular phosphatase activity by the phosphatase inhibitor microcystin LR or the ATP analogue ATP gamma S. This finding strongly suggests the presence of a phosphorylation event in the calcium-dependent activation pathway for both currents. 4. Intracellular dialysis with peptide inhibitors of the multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaM kinase) blocked the activation of both the chloride and cation conductances by elevated [Ca2+]i. Dialysis with an inactive control peptide had no effect on the activation of either current. CaM kinase thus appears to be critically involved in the calcium-dependent activation of both the chloride and cation currents in these cells. 5. Associated with the whole-cell cation conductance were macroscopic tail currents observed at the chloride reversal potential. The distinct kinetic properties of these tail currents were used as a biophysical 'signature' of the whole-cell conductance. 6. In excised, inside-out membrane patches, [Ca2+]i activated single cation channel activity. These channels had a mean conductance of 20 pS, were impermeable to NMDG, and their mean open probability increased at positive membrane potentials. The properties of these single channel events thus closely resemble those reported previously for calcium-activated cation channels in epithelia. 7. Using a novel 'tail current' voltage clamp protocol in excised membrane patches, we observed that ensemble averages of single cation channel events reproduced the behaviour and kinetic properties of the macroscopic tail currents of the calcium-activated cation conductance. This finding provides evidence that the observed single channel events probably underlie the macroscopic cation current recorded from intact cells. 8. The results from this study demonstrate that CaM kinase mediates the calcium-dependent activation of both a chloride and a non-selective cation current in human T84 epithelial cells. Using single channel recordings, we believe we have identified the corresponding whole-cell current for the 20-40 pS calcium-activated cation channel activity reported previously in epithelia and other cell preparations. Physiologically, a calcium-activated inward cation current would allow sodium influx in association with calcium-dependent electrolyte and protein secretion. Thus CaM kinase-dependent activation of cation channels may serve as a co-ordinated influx pathway to balance the efflux and influx of osmotically active solutes as part of an overall cell volume regulatory mechanism.

Amino Acid Sequence↗