phosphorus-radioisotopes and tetraphenylphosphonium

The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation-magic-angle spinning nuclear magnetic resonance spectroscopy (CP-MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP-MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

Topics: Amino Acid Sequence; Antiporters; Bacterial Proteins; Escherichia coli Proteins; Ions; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Molecular Structure; Onium Compounds; Organophosphorus Compounds; Phosphorus Radioisotopes

Antiphospholipid antibodies (aPL) are associated with neurological diseases such as stroke, migraine, epilepsy and dementia and are thus associated with both vascular and non-vascular neurological disease. We have therefore examined the possibility that these antibodies interact directly with neuronal tissue by studying the electrophysiological effects of aPL on a brain synaptosoneurosome preparation. IgG from patients with high levels of aPL and neurological involvement was purified by protein-G affinity chromatography as was control IgG pooled from ten sera with low levels of aPL. Synaptoneurosomes were purified from perfused rat brain stem. IgG from the patient with the highest level of aPL at a concentration equivalent to 1:5 serum dilution caused significant depolarization of the synaptoneurosomes as determined by accumulation of the lipophylic cation [3H]-tetraphenylphosphonium. IgG from this patient as well as IgG from two elderly patients with high levels of aPL were subsequently shown to permeabilize the synaptosomes to labeled nicotinamide adenine dinucleotide (NAD) and pertussis toxin-ADP-ribose transferase (PTX-A protein) as assayed by labeled ADP-ribosylation of G-proteins in the membranes. No such effects were seen with the control IgG. aPL may thus have the potential to disrupt neuronal function by direct action on nerve terminals. These results may explain some of the non-thromboembolic CNS manifestations of the antiphospholipid syndrome.

Topics: Adult; Aged; Animals; Antibodies, Antiphospholipid; beta 2-Glycoprotein I; Brain; Cell Membrane; Cell Membrane Permeability; Dose-Response Relationship, Immunologic; Electrophysiology; Female; Glycoproteins; Humans; Immunoglobulin G; Membrane Potentials; Middle Aged; Neurons; Onium Compounds; Organophosphorus Compounds; Pertussis Toxin; Phosphorus Radioisotopes; Poly(ADP-ribose) Polymerases; Rats; Synaptosomes; Tritium; Virulence Factors, Bordetella

Lipophilic ions modify the affinity of the cation binding sites of the membrane-bound Na,K-ATPase. We studied the effect of the lipophilic ions tetraphenyl-phosphonium (TPP+) and tetraphenylboron (TPB-) on the binding of Na+ and K+ to the cation site(s) that are exhibited by the enzyme during the catalytic cycle: the high-affinity (inside) Na-binding site, site I, the low-affinity (outside) Na-leaving site, site II, and the high-affinity (outside) K-site, site III. Site I: In the presence of TPP+ (positive charge added to the lipid environment) a higher Na(+)-concentration was needed to obtain phosphorylation of the enzyme, whereas in the presence of TPB- (negative charge added to the lipid environment) phosphorylation was obtained at a lower Na(+)-concentration, but the change in apparent K0.5 for Na+ was small, (K0.5Na,TPP = 0.180 mM and K0.5Na,TPB = 0.07 mM), indicating only a minor influence of membrane charge on Na+ binding to site I. Site II: Compared to control conditions, more Na+ was required to inhibit ATP-hydrolysis and to increase the steady-state level of ADP-sensitive phosphoenzyme when TPP+ was present, and the opposite was observed with TPB-, indicating a strong influence of membrane charge on the Na+ occupancy of site II. Site III: TPP+ induced a significant decrease both in the rate of K-dependent dephosphorylation of preformed E32P and in the K+ affinity. The effect of TPP+ on the ATP hydrolysis rate strongly resembled the effect of decreasing [KCl]. The results indicated a pronounced effect of adding positive charge to the lipid environment of site III.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Diphosphate; Animals; Binding Sites; Cattle; Hydrolysis; In Vitro Techniques; Kinetics; Membranes; Onium Compounds; Organophosphorus Compounds; Phosphorus Radioisotopes; Phosphorylation; Potassium Chloride; Sodium; Sodium-Potassium-Exchanging ATPase; Tetraphenylborate