URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit.

The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

Impaired mitochondrial beta-oxidation in a patient with an abnormality of the respiratory chain. Studies in skeletal muscle mitochondria.

Defects of complex I of the mitochondrial respiratory chain are important causes of neurological disease. We report studies that demonstrate a severe deficiency of complex I activity with less severe abnormalities of complexes III and IV (less than 5, 63, and 30% of control values, respectively) in a skeletal muscle mitochondrial fraction from a 22-yr-old female with weakness, lactic acidemia, and the deposition of intramuscular neutral lipid. The observation that lipid accumulates in this and other patients with complex I deficiency suggests impaired mitochondrial fatty acid oxidation. To investigate this mechanism we have shown impaired flux through beta-oxidation [( U-14C]hexadecanoate oxidation was 66% of control rate) and accumulation of specific acyl-CoA ester intermediates. The changes in fatty acid metabolism in complex I deficiency are secondary to the reduced state within the mitochondrial matrix with low NAD+/NADH ratios.

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone.

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.

A spin label study of the lipid boundary layer of mitochondrial NADH-ubiquinone oxidoreductase.

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I) is a lipoprotein enzyme containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin. Enzyme preparations containing endogenous cardiolipin and a range of either soyabean PC or dimyristoylphosphatidylcholine (DMPC) concentrations have been made. Using a spin-labelled fatty acid, two probe environments differing in mobility have been shown to be present. The fatty acid probe has a relative binding constant (or partition coefficient between lipid and protein) of unity. The boundary layer or lipid annulus reported by the probe has a value of approx. 300 lipid molecules per molecule of enzyme FMN in preparations containing soyabean PC, or DMPC above the phase transition temperature of the latter. In soyabean PC-replaced enzyme the apparent size of the boundary layer is independent of temperature between 30 degrees C and 14 degrees C but shows a modest increase to about 400 lipid molecules per molecule of FMN between 14 degrees C and 2 degrees C. Complex I replaced with high concentrations of DMPC gives non-linear Arrhenius plots of NADH-ubiquinone oxidoreductase activity. The results of the ESR experiments show that both boundary layer and bulk lipid must be motionally restricted for this to occur. Thus, the change in activity is probably not caused by an effect exerted directly on the catalytic activity of the enzyme but is more likely due to restriction of free diffusion of ubiquinone to its site of reduction.

Biosynthesis and import into the mitochondrion of L-3-glycerophosphate dehydrogenase, and the effect of thyroid hormone deficiency on gene expression.

Mitochondrial L-3-glycerophosphate dehydrogenase (EC 1.1.99.5) is synthesised in bovine kidney (NBL-1) cells treated with uncoupler as a cytosolic precursor with Mr = 76,000 indistinguishable from the mature form. In vitro translation of rat liver mRNA also gives rise to a product of Mr = 76,000 but when this is imported into mitochondria it is processed to a product of Mr = 66,000. L-3-Glycerophosphate dehydrogenase activity and immunoreactive protein are greatly decreased in liver mitochondria from hypothyroid rats. Paradoxically, in vitro translation of the mRNA from such animals gives rise to large amounts of the protein, much greater than that synthesised from euthyroid mRNA and comparable with that produced from hyperthyroid mRNA.

The effects of lipid fluidity on the rotational diffusion of complex I and complex III in reconstituted NADH-cytochrome c oxidoreductase.

NADH-ubiquinone oxidoreductase (Complex I) can be recombined with ubiquinol-cytochrome c oxidoreductase (Complex III) to reconstitute NADH-cytochrome c oxidoreductase. Two modes of interaction have been found. In one, the Complexes interact stoichiometrically in one to one molar ratios to give a binary Complex I-III unit. In the other, the kinetics of NADH-cytochrome c oxidoreductase are characteristic of 'Q-pool' behaviour seen in intact mitochondria and submitochondrial particles in which the Complexes need not interact directly but can do so via a pool of mobile ubiquinone. Stoichiometric behaviour is found when only boundary layer or annular lipid is present or the lipid is in the gel phase. The lipid is immobile on the ESR time scale and protein rotational diffusion, measured by saturation transfer ESR, is very slow. Q-pool behaviour is found when mobile extra-annular lipid phase is also present. Protein rotational diffusion is rapid and characteristic of a fully disaggregated state. We have also used freeze-fracture electron microscopy of reconstituted NADH-cytochrome c oxidoreductase to monitor protein aggregation and lateral phase separation of lipids and proteins under various conditions. We discuss our findings in relation to models for lateral interactions between respiratory chain enzymes.

Lithium and the phosphoinositide cycle: an example of uncompetitive inhibition and its pharmacological consequences.

The ability of lithium to exert profound and selective psychopharmacological effects to ameliorate manic-depressive psychosis has been the focus of considerable research effort. There is increasing evidence that lithium exerts its therapeutic action by interfering with polyphosphoinositide metabolism in brain and prevention of inositol recycling by an uncompetitive inhibition of inositol monophosphatase. Stefan Nahorski, Ian Ragan and John Challiss discuss this unusual stimulus-dependent form of enzyme inhibition, emphasizing that the selectivity exhibited by lithium depends upon the degree of inositol lipid hydrolysis and polyphosphoinositide dephosphorylation.

Schizophrenia and L-745,870, a novel dopamine D4 receptor antagonist.

The discovery of a novel high-affinity and selective dopamine D4 receptor antagonist, L-745,870, and the results of clinical trials with this compound are reviewed. Despite several lines of evidence which suggest that a selective D4 receptor antagonist may be an effective antipsychotic agent with a lower propensity to induce extrapyramidal side-effects, L-745,870 was ineffective as an antipsychotic in humans.

The effects of substrates, products and other ligands on the susceptibility of inositol monophosphatase to proteolysis by endoprotease lys-C.

Inositol monophosphatase is cleaved by endoprotease lys-C at a single site (Lys36-Ser37). The rate of proteolysis is greatly reduced in the presence of substrate (D,L-Ins(1)P) and Mg2+, and less so in the presence of Pi and Mg2+, consistent with protection of the susceptible bond in the E-P or E-Pi states of the enzyme. Potentiation by Li+ of the protection afforded by a substrate analogue, 1S-phosphoryloxy-2R,4S-dihydroxycyclohexane, and Mg2+ supports the idea that Li+ binds to the E-P state.

Immunological characterization of the mitochondrial 2-oxoglutarate carrier from liver and heart. Organ specificity.

Antibodies have been prepared against the 2-oxoglutarate transport proteins purified from bovine heart and rat liver mitochondria. The anti-heart antiserum cross-reacts with the 2-oxoglutarate carrier (OGC) from beef, pig, rat and rabbit heart, but not with the OGC from liver of the same animals. Conversely, the anti-liver antiserum recognizes the carrier protein from liver of all species tested but not from heart. Immunoinactivation of oxoglutarate transport activity by the antibodies is also tissue specific. Peptide maps of purified OGC show structural differences between the carrier from heart and liver of the same animal species. These results indicate the existence of isoforms of the OGC in heart and liver.

Structural relationships between the NADH dehydrogenases of Paracoccus denitrificans and bovine heart mitochondria as revealed by immunological cross-reactivities.

An antibody raised against two subunits (Mr 48 000 and 25 000) of NADH dehydrogenase from Paracoccus denitrificans cross-reacts with one of more than 20 polypeptides that form the bovine heart mitochondrial NADH dehydrogenase. The cross-reacting subunit has Mr 51 000 and is believed to be the NADH-binding subunit of the enzyme. Antibodies raised against certain subunits of the bovine heart NADH dehydrogenase were tested for cross-reactivity with P. denitrificans cytoplasmic membranes. Of those tested, only one, an antibody specific for the 49 kDa subunit of mitochondrial NADH dehydrogenase, cross-reacted with the bacterial membranes. It recognised a polypeptide of approximate Mr 46 000. This is an indication for a previously undetected third subunit of NADH dehydrogenase from P. denitrificans. The immunological cross-reactions indicate that the NADH dehydrogenases from P. denitrificans and bovine heart mitochondria are related structurally.

Bovine inositol monophosphatase: proteolysis and structural studies.

Bovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of alpha helices. The most susceptible bond (Lys-36--Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Garnier et al. [(1978) J. Mol. Biol. 120, 97-120].

Bovine inositol monophosphatase. The identification of a histidine residue reactive to diethylpyrocarbonate.

The inositol monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217-->Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme.

[3H]L-655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit.

A compound (L-655,708) has been identified which has at least 50-fold selectivity for the benzodiazepine site on GABAA receptors containing an alpha 5 subunit over those containing an alpha 1, alpha 2, alpha 3 or alpha 6 subunit in combination with beta 3 and gamma 2. The compound was radiolabelled with tritium and investigated as a novel radioligand which recognizes the benzodiazepine site of GABAA receptors which contain the alpha 5 subunit. [3H]L-655,708 labels one saturable and specific population of binding sites in rat hippocampus with a Kd of 2.4 +/- 0.7 nM and a Bmax of 256 +/- 42 fmol/mg protein. The pharmacology of the binding site labelled was consistent with that of receptors present in cells transfected with alpha 5, beta 2 and gamma 2 and with receptors immunoprecipitated from rat brain with an alpha 5-selective antiserum. It is concluded that [3H]L-655,708 is the first radioligand to date which is selective for any BZ2 subtype of the GABAA receptor and should provide a valuable tool for elucidating the structure and function of the alpha 5-containing GABAA receptor subtype.

4-Heterocyclylpiperidines as selective high-affinity ligands at the human dopamine D4 receptor.

5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with > 500-fold selectivity over hD2 and > 200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.

Characterization of the effects of lithium on phosphatidylinositol (PI) cycle activity in human muscarinic m1 receptor-transfected CHO cells.

1. The effects of lithium on [3H]-inositol and [3H]-cytidine incorporation into [3H]-inositol monophosphates ([3H]-InsP1) and [3H]-cytidine monophosphorylphosphatidate ([3H]-CMP-PA), respectively, and inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) mass were studied in carbachol-stimulated human m1 muscarinic receptor-transfected Chinese hamster ovary cells (m1 CHO cells). 2. Lithium alone (10 mM) had no appreciable effects on any of the four parameters measured; it was only in carbachol-stimulated cells that the effects of lithium became apparent. 3. In the presence of carbachol (1 mM), lithium (10 mM) caused a relatively rapid (within 5 min) accumulation of [3H]-InsP1 and [3H]-CMP-PA which continued up to about 20-30 min, after which accumulation slowed down. On the other hand, the elevation in InsP3 and InsP4 levels produced by carbachol was not altered by lithium in the short-term and only at later times (> 20-30 min) was the response attenuated, with InsP3 and InsP4 levels approaching basal. 4. The effects of lithium on carbachol-stimulated [3H]-InsP1 and [3H]-CMP-PA accumulation and the attenuation of the carbachol-induced elevation of InsP3 and InsP4 were all dose-dependent, with EC50s in the region of 1 mM. 5. The lithium-induced effects on [3H]-CMP-PA and InsP3 and InsP4 in carbachol-stimulated cells could be reversed, in a dose-dependent manner, by preincubation with exogenous myo-inositol (EC50 = 2-3 mM) but not by the inactive analogue scyllo-inositol, indicating that these effects occur as a consequence of depletion of inositol. 6. The temporal effects of lithium are consistent with lithium inhibiting inositol monophosphatase,causing accumulation of InsP1, resulting in lower free inositol levels. This leads to accumulation of CMP-PA and reduced PI synthesis which, once agonist-linked membrane inositol phospholipids are depleted, produces attenuated InsP3 and InsP4 responses.7. These results in ml CHO cells support the hypothesis that lithium affects the PI cycle cell signalling pathway by depletion of inositol due to inhibition of inositol monophosphatase.

Relationship between inhibition of cyclic AMP production in Chinese hamster ovary cells expressing the rat D2(444) receptor and antagonist/agonist binding ratios.

1. Radioligand binding assays using [3H]-(-)-sulpiride, in the presence of 1 mM ethylenediaminetetraacetic acid (EDTA) and 100 microM guanylylimidodiphosphate (GppNHp) and [3H]-N0437 were developed to label the low and high agonist affinity states of the rD2(444) receptor (long form of the rat D2 receptor) respectively. The ratios of the affinities of compounds in these two assays (Kapp [3H]-(-)-supiride/Kapp [3H]-N-0437) were then calculated. 2. The prediction that the binding ratio reflected the functional efficacy of a compound was supported by measurement of the ability of a number of compounds acting at dopamine receptors to inhibit rD2(444)-mediated inhibition of cyclic AMP production. When the rank order of the ratios of a number of these compounds was compared to their ability to inhibit the production of cyclic AMP, a significant correlation was seen (Spearman rank correlation coefficient = 0.943, P = 0.01). 3. In conclusion, the sulpiride/N-0437 binding ratio reliably predicted the efficacy of compounds acting at dopamine receptors to inhibit cyclic AMP production mediated by the rD2(444) receptor.