H.G.P. Swarts

NDUFA2 Complex I Mutation Leads to Leigh Disease

Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5. Screening of this gene on genomic DNA revealed a mutation that interferes with correct splicing and results in the skipping of exon 2. Exon skipping was confirmed on the mRNA level. The mutation in this accessory subunit causes reduced activity and disturbed assembly of complex I. Furthermore, the mutation is associated with a mitochondrial depolarization. The expression and activity of complex I and the depolarization was (partially) rescued with a baculovirus system expressing the NDUFA2 gene.

Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the α- and β-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 μM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.

Cation fluxes and Na+-K+-activated ATPase activity in erythrocytes of patients with essential hypertension.

SUMMARY Recently it has been claimed that the active potassium influx in erythrocytes of patients with essential hypertension would be increased. In view of the diagnostic and possibly therapeutic potential of this claim, we have determined the Na+-K+ activated ATPase activity and the affinity of the enzyme for Na+, K+, and ATP in membranes isolated from erythrocytes of hypertensive (with and without medication) and normotensive subjects. Subsequently, the active (ouabain-sensitive) sodium and potassium fluxes and their ratios have been determined after treatment of intact erythrocytes either with cold or with p-chloromercuribenzenesulfonate (PCMBS). Finally, in view of a subsequent daim that the furosemide-sensitive, ouabain-insensitive cation fluxes would be greatly reduced in erythrocytes of patients with essential hypertension, we have determined these fluxes in cboline chloride medium containing ouabain with and without furosemide. For none of these parameters has any significant difference between hypertensive and normotensive subjects been found except for a decrease in the ouabain-sensitive K* influx after cold treatment in hypertensives. This is also true for the hypertensive.subjects who had a known hypertensive parent. It is concluded that the results do not support a role of Na+-K+ activated ATPase or the furosemide-sensitive cation carrier in the pathogenesis of essential hypertension, and that ouabain-sensitive and furosemide-sensitive cation flux determinations in erythrocytes do not seem to be useful for the diagnosis of this condition.

Functional expression of gastric H,K-ATPase using the baculovirus expression system

A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α‐ as well as the β‐subunit of the gastric H.K‐ATPase. Sf9 cells infected with this virus exhibited a K+‐ and SCH 28080‐sensitive ATP‐dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K‐ATPase. This activity was not present in control membranes containing only one of the two H,K‐ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K‐ATPase.

Kinetics of Cu2+ inhibition of Na+K+-ATPase

The interaction of Cu2+ with enzymatic activity of rabbit kidney Na+/K(+)-ATPase was studied in media with buffered, defined free Cu2+ levels. The IC50-values are 0.1 mumol/l for Na+/K(+)-ATPase and 1 mumol/l for K(+)-pNPPase. Dithiothreitol (DTT) reverses the inhibitory effect of Cu2+ in vitro. Cu2+ exerts non-competitive effects on the enzyme with respect to Na+, K+, ATP or pNPP, but has a mixed-type inhibitory effect with respect to Mg2+. It is concluded that the appreciation of the inhibitory effect of Cu2+ on this enzyme requires carefully composed assay media that include a buffer for Cu2+, and that the IC50-values calculated according to this model indicate that Cu2+ may be more toxic than previously anticipated.

Role of calcium in exocrine pancreatic secretion. II.Comparison of the effects of carbachol and the ionophore A-23187 on enzyme secretion and calcium movements in rabbit pancreas

1. The secretory effects of carbachol and the ionophore A-23187 on the isolated rabbit pancreas and rabbit pancreas fragments are compared in order to obtain more insight in the involvement of calcium in the stimulus-secretion coupling of pancreatic enzyme secretion. 2. The divalent cation ionophore A-23187 mimicks the effect of carbachol on pancreatic enzyme secretion in both preparations. 3. The action of the ionophore is dependent on the presence of extracellular calcium. The carbachol effect is much less dependent on calcium, as it occurs even in a Ca2+ -free medium containing 10(-4) M ethyleneglycol-bis(beta-aminoethylether)-N,N-tetraacetic acid. 4. Carbachol causes a marked increase in the 45Ca2+ efflux from pre-loaded pancreas fragments in both a normal Krebs-Ringer bicarbonate medium and this Ca2+ -free medium. 5. Although the tissue still contains about 50% of its original 45Ca2+ content, at the time of stimulation, the ionophore has little or no effect on the 45Ca2+ efflux. This indicates that the cytoplasmic 45Ca2+ concentration is very low, and hence that most of the 45Ca2+ must be sequestered in one or more intracellular stores. 6. It is concluded that both substances stimulate pancreatic enzyme secretion by increasing the cytoplasmic calcium concentration, through an increase in the calcium permeability of the plasma membrane in the case of the ionophore, and through a release of Ca2+ from intracellular stores in the case of carbachol.

Regulation of expression of Na+, K+-ATPase in androgen-dependent and androgen-independent prostate cancer

SummaryThe β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent.

Studies on (Na+ + K+)-activated ATPase. XLIV. Single phosphate incorporation during dual phosphorylation by inorganic phosphate and adenosine triphosphate.

(Na+ + K+)-ATPase can be phosphorylated by its substrate ATP as well as by its product inorganic phosphate. The maximal capacity for phosphorylation by either of these two substances is one mol phosphate per mol enzyme. In order to investigate whether the enzyme molecule possesses only on phosphorylation site common to ATP and Pi, or two phosphorylation sites, one for ATP and one for Pi, dual phosphorylation of the enzyme has been carried out. Under conditions, which are maximally favourable for each type of phosphorylation, successive phosphorylation by Pi and ATP leads to a maximal incorporation of only one mol phosphate per mol enzyme. The phosphorylation capacity for ATP decreases by the same amount as the Pi-phosphorylation level increases, without an effect on the apparent affinity for ATP. The results can be explained by assuming either a single common phosphorylation site for Pi and ATP, or a conformational change of the enzyme following phosphorylation by Pi, which excludes phosphorylation by ATP.

Role of calcium in exocrine pancreatic secretion I. Calcium movements in the rabbit pancreas

1. Calcium movements in the isolated rabbit pancreas and in rabbit pancreas fragments have been studied with the aid of 4 5 Ca2+. 2. Addition of 4 5 Ca2+ to the incubation medium of the isolated rabbit pancreas results in an immediate appearance of isotope in the secreted fluid reaching a constant specific activity in 30 min. The absolute activity in the secreted fluid is 30-40% of that in the incubation medium. 3. Addition of 10(-5) M carbachol after 2 h preincubation with 4 5 Ca2+ results in enzyme secretion accompanied by calcium release. There is also an increase in 4 5 Ca2+ secretion, but this is maximal 10 min after the protein and total calcium peaks. 4. Partial removal of 4 5 Ca2+ from the bathing medium, before stimulation, reduces the increase in 4 5 Ca2+ secretion nearly proportionally. 5. [3H]Mannitol, added to the bathing medium, appears in the secreted fluid and behaves upon carbachol stimulation similarly to 4 5 Ca2+. 6. Upon repeated stimulation with 10(-5) M acetylcholine, a 4 5 Ca2+ peak appears, even in virtual absence of enzyme secretion. In this case the peak coincides with a small total calcium peak. 7. Efflux studies of rabbit pancreas fragments, preloaded with 4 5 Ca2+, show a carbachol-stimulated 4 5 Ca2+ efflux in addition to a release of amylase. 8. These studies indicate that there are three calcium movements in rabbit pancreas which can all be influenced by cholinergic agents: (a) an extracellular route for calcium and other small molecules and ions; (b) a calcium release across the apical membrane along with the enzymes, originating from a pool which does not freely exchange with 4 5 Ca2+ in the bath; (c) a calcium flux across the serosal membrane, which involves calcium exchanging freely with 4 5 Ca2+ from the bath. The third flux is thought to result from an increase in cytoplasmic calcium, which may be involved in the stimulus-secretion coupling of pancreatic enzyme secretion.

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase☆

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.