Abnormal glyceraldehyde-3-phosphate dehydrogenase binding and glycolytic flux in Autosomal Dominant Polycystic Kidney Disease after a mild oxidative stress.

AIM: The aim of this study was, a) to investigate the effect of mild oxidative stress on glycolytic flux and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binding in erythrocytes from patients with autosomal dominant polycystic kidney disease (ADPKD), and b) to examine whether the modulation of GAPDH-binding to the red cell membrane leads to changes in glycolytic flux. PATIENTS AND METHODS: The rate of lactate production in intact erythrocytes and the GAPDH/actin ratio in erythrocyte ghost membranes were measured before and after treating cells with t-butyl hydroperoxide or N-ethylmaleimide (NEM) in 13 ADPKD patients and 12 controls. RESULTS: t-bytyl hydro-peroxide had a significant effect on both lactate production and GAPDH/actin ratio in healthy subjects, but it had essentially no effect on ADPKD patients in which both parameters already resembled those of the peroxide-treated controls. NEM treatment after 300 sec had a very significant effect on both lactate production and GAPDH/actin ratio in both patient and control cells. However, after 10 sec the effect on GAPDH/actin ratio was only significant in the erythrocytes of ADPKD patients. In every experiment glycolytic lactate production correlated negatively with membrane-bound GAPDH/actin ratio. CONCLUSIONS: We conclude that glycolytic flux and GAPDH binding in erythrocytes from ADPKD patients respond abnormally to both a mild oxidative stress and brief exposure to NEM.

Formulation of a coupled mechanism between solute diffusion, phosphatase-kinase reactions and membrane potentials for the primary active transport of phosphorylated substrates through biological membranes.

Coupled interrelations occurring between a phosphatase/kinase reaction sequence acting in unstirred layers and on both sides of a charged biomembrane pore structure are presented as a plausible kinetic model for the primary active transport of phosphorylated molecules. Simulations conducted at the cell level and with credible numerical values demonstrate that the enzymes positions strongly regulate the membrane permeability for the transported substrate. Depending on both the enzymes positions (more or less far from the membrane) and the membrane charges, the membrane may appear either impervious, either permeable or able to actively transport a phosphorylated substrate. Globally all happens as if, in function of the enzymes positions, a permanent pore may be regulated, changing from a more closed to a more open conformation.

Abnormal thiol reactivity of tropomyosin in essential hypertension and its association with abnormal sodium-lithium countertransport kinetics.

OBJECTIVES: To identify a thiol protein that is abnormal in a subgroup of essential hypertensive (EHT) patients who have a strong family history of hypertension and cardiovascular disease and have a low Km of erythrocyte Na/Li countertransport (CT). METHODS: To detect biotin maleimide labelling of a key thiol protein to investigate its reaction with N-ethylmaleimide (NEM) in normal and EHT erythrocytes. RESULTS: The thiol protein of 33 kDa apparent molecular weight (p33) identified by the loss of labelling with biotin maleimide was identified as tropomyosin due to its retarded running in 6 mol/l urea gels and immunoblotting. The NEM reaction with p33 detected by loss of subsequent biotin maleimide labelling is biphasic in normal control erythrocytes with the rate in the first 30 s double that after 30 s. In EHT erythrocytes NEM reaction (1) after 30 s is faster than normal and (2) in the first 30 s causes a paradoxical increase in apparent biotin maleimide labelling. In normal control erythrocytes, the loss of biotin maleimide labelling with NEM reaction or the faster phenylmaleimide reaction follows the same time course as the decrease in Km of Na/Li CT. CONCLUSIONS: NEM reaction with p33 requires two thiols. Only the cytoskeletal form of tropomyosin from the TM3 gene has more than one thiol group and agrees with SDS-PAGE mobility. Tropomyosin is a strong candidate to explain the familial abnormality in EHT with abnormal Na/ Li CT and it could explain many of the characteristics of this disease.

Radicals and oxidative stress in diabetes.

Recent evidence is reviewed indicating increased oxidative damage in Type 1 and Type 2 diabetes mellitus as well as deficits in antioxidant defence enzymes and vitamins. Mechanisms are considered whereby hyperglycaemia can increase oxidative stress, and change the redox potential of glutathione and whereby reactive oxygen species can cause hyperglycaemia. It is argued that oxygen, antioxidant defences, and cellular redox status should now be regarded as central players in diabetes and the metabolic syndrome.

Erythrocyte membrane thiol proteins associated with changes in the kinetics of Na/Li countertransport: a possible molecular explanation of changes in disease.

BACKGROUND: Abnormal erythrocyte Na/Li countertransport is associated with diseases such as essential hypertension and diabetic renal disease. Although it seems unlikely that Na/Li countertransport contributes to any disease process, it may be abnormal because of a change in the cell membrane that is part of the disease process. METHODS: We have shown that Na/Li countertransport kinetics are modified by two types of thiol group. One of these, which we have called 'type 1', is rapidly alkylated by N-ethylmaleimide to give a kinetic pattern similar to that in the above diseases. RESULTS: AtpH 6 and 2 degrees C, both N-ethylmaleimide and iodoacetamide cause the K(m) of Na/Li countertransport to decrease to completion in 300s, with 78% (SEM 6%) of the decrease occurring in 30s. Using these reaction conditions, N-ethylmaleimide reacted with a unique thiol group on a 33-kD protein, blocking its subsequent reaction with biotin maleimide. This 33-kD protein was present in rabbit erythrocytes, which have high levels of Na/Li countertransport, but absent from rat erythrocytes, which have no Na/Li countertransport. Iodoacetyl biotin labelled a 60-kD protein that was specifically blocked by iodoacetamide. CONCLUSION: We suggest that these proteins are members of a cluster of membrane proteins that can modify Na/Li countertransport and may have a functional role in the disease processes.

Sodium-lithium countertransport: physiology and function.

Current opinions on the relationships between erythrocyte sodium-lithium countertransport kinetics and primary hypertension, hyperlipidaemia and diabetic nephropathy are reviewed. Problems associated with the assay are analysed. Some possible mechanisms that could modify the kinetics of ion exchange are examined. The question of what catalyses sodium-lithium countertransport is discussed, but not answered. Some models are put forward showing how a study of sodium-lithium countertransport kinetics could further our understanding of important disease processes.

Thiol group control of sodium-lithium countertransport kinetics in uraemia: evidence of a membrane abnormality affected by haemodialysis.

Uraemia affects erythrocyte metabolism and membrane function but no consistent effect on Na/Li countertransport (CT) has been reported. We report only small differences in Na/Li CT at 150 mmol/l Na over haemodialysis, but major differences in other properties of Na/Li CT. The Km for external sodium and Vmax both increased during haemodialysis but the Vmax/Km ratio, which was greater than normal, was not affected. The thiol reagent, N-ethylmaleimide (NEM), which causes a decrease in Km and Vmax in normal subjects, had no effect on Km in the predialysis erythrocytes. After haemodialysis, the sensitivity of Na/Li CT to NEM was improved. The changes in Na/Li CT kinetics were not related to changes in membrane lipid fluidity or plasma lipids. These observations suggest that uraemia affects a thiol group that controls Na/Li CT kinetics and that haemodialysis temporarily improves this aspect of membrane function.

Modification of erythrocyte Na+/Li+ countertransport kinetics by two types of thiol group.

Erythrocyte Na+/Li+ countertransport activity is decreased by reagents that react with thiol groups. An understanding of the role of these groups in control of Na+/Li+ countertransport may help to explain its association with disease states. The effect of thiol reactive agents on the kinetic parameters of Na+/Li+ countertransport has not previously been described. In choline medium, N-ethylmaleimide (NEM) and iodoacetamide (IAamide) cause a rapid decrease of about 40% in Km for external sodium (Km(So)) that is complete in 10 s with a much smaller change in Vmax and an increase in the Vmax/Km ratio. In Na medium, NEM and IAamide both cause a rapid decrease in Km(So) and Vmax. With NEM the partial reduction in Vmax is complete in 100s although the NEM is sufficient to reduce Vmax up to 15 min. With IAamide the decrease in Vmax is initially slower but it continues apparently towards complete inhibition. These results indicate at least two types of thiol group controlling Na+/Li+ countertransport kinetics. The type 1 thiol reaction is Na independent and causes an increase in the apparent rate constant for Na association with the unloaded carrier so that Vmax/Km rises and Km(So) decreases. The type 2 thiol reaction is facilitated by Na at the outside ion-binding site and causes a decrease in Vmax, possibly by total blockage of carriers with IAamide but by a different mechanism with NEM such as reduced turnover rate.

Sulphydryl group control of sodium-lithium countertransport kinetics: a membrane protein control abnormality in essential hypertension.

Erythrocyte sodium-lithium countertransport (SLC) is an obligatorily coupled equimolar exchange of intracellular sodium or lithium with extracellular sodium or lithium. SLC is partially inhibited by N-ethylmaleimide (NEM) but only when a transported ion (sodium of lithium) is present in the extracellular medium. In essential hypertensive patients with a strong family history of hypertension the Km of SLC for extracellular sodium was lower and Vmax tended to be higher than in normal controls, but the ratio Vmax/Km gave a much clearer distinction between the two groups. After NEM treatment, the remaining SLC activity in normal individuals had a lower Vmax and Km for sodium but Vmax/Km was not affected. In essential hypertensives the remaining SLC activity after NEM again had lowered Vmax and Km but in these patients the Vmax/Km was much lower than in untreated erythrocytes and was then the same as in normal controls. On the assumption that NEM reacts with a -SH group on a membrane protein that regulates SLC, and that the ratio Vmax/Km reflects a rate constant for binding extracellular sodium to the unloaded carrier, the results suggest that (a) essential hypertensives have an increased rate of sodium binding to the transporter and (b) this is due to abnormal behaviour of a membrane -SH group.

Redox-linked proton translocation by direct-coupled ligand conduction.

The term "direct-coupled" is considered in the context of redox-linked proton translocation mechanisms, and the origins of this concept, its philosophical implications, applications, and contributions to the development of bioenergetics, are discussed.

What determines the substrate specificity of the multi-drug-resistance pump?

Multi-drug-resistance protein (P-glycoprotein) turns out to be an ATP-hydrolysing transmembrane pump that increases the resistance of cells in which it is expressed by actively extruding toxic chemicals. The baffling question is how does the pump know which chemicals to extrude? Common features among its substrates are still elusive. The question raised here concerns the relationship between this pump and that known for many years as capable of extruding glutathionyl and cysteinyl S-conjugates of xenobiotics. Are excreted drugs conjugated before excretion? Does the multi-drug-resistance pump recognize a simple chemical tag put on xenobiotics by a family of transferase enzymes?

Triphasic reduction of bH and the absence of equilibration at the i-site of bc1 complex.

The concept of a stabilized semiquinone radical forming the basis of a two-electron gate has long been familiar in the context of the quinone-reducing site in photosynthetic systems and has already been suggested to play a role at the i-site of the bc1-type complexes. It is here pointed out that this concept is sufficient to explain the so-called triphasic reduction kinetics of cytochrome bH, in which cytochrome bH goes partially reduced, is reoxidized and then goes fully reduced. The rate constants for binding and unbinding of quinone, quinol and semiquinone at the i-site are discussed, and a kinetic model featuring slow release of the i-site semiquinone is shown to display many features of the kinetics of electron transfer at the i-site.

The location of CuA in mammalian cytochrome c oxidase.

Imposition of a protonmotive force across the inner membrane of coupled cyanide-inhibited, beef heart mitochondria by addition of ATP causes reduction of cytochrome c and CuA with concomitant oxidation of haem aA. The data are consistent with previous demonstrations of an intramembrane location of haem aA but further indicate that CuA is very close to the cytosolic surface of the membrane. The implications of this finding for electron transfer route and the site of the proton pumping chemistry are discussed.

Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol.

The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol. A transmembrane electric potential difference was then developed, either by electron flow from [Ru(NH3)6]Cl2 to oxygen or by ATP hydrolysis. The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566. No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems.

Protonmotive stoichiometry of rat liver cytochrome c oxidase: determination by a new rate/pulse method.

The stoichoimetry of vectorial H+ ejection coupled to electron flow through the cytochrome c oxidase (EC 1.9.3.1) of rat liver mitochondria was determined by a new rate/pulse method. This is a modification of the oxygen-pulse method. Electron flow through the oxidase is initiated by adding oxygen to suspensions of anaerobic mitochondria at a known and constant rate. Cytochrome c oxidase was examined directly or in combination with cytochrome c reductase (ubiquinol:ferricytochrome c oxidoreductase). In both cases the----H0+/2e- ratio was found to be constant during the time-course of oxygen reduction, and thus independent of delta pH. The stoichiometries observed were consistent with mechanistic stoichiometries of 2 and 6 for cytochrome c oxidase alone and cytochrome c oxidase together with cytochrome c reductase, respectively. The stoichiometry of cytochrome c reductase alone was also examined, by using ferricyanide in place of oxygen. The results obtained were consistent with the accepted mechanistic stoichiometry of 4 for this enzyme.