The oxoglutarate/malate carrier of rat brain mitochondria operates by a uniport exchange mechanism.

Here, the oxoglutarate carrier, already isolated from various sources and described in the literature, has been purified from rat brain and reconstituted in proteoliposomes for an accurate kinetic study. The rate of uptake of labelled oxoglutarate and malate has been measured in various conditions, essentially in double substrate experiments. The data so obtained fit the hypothesis that the carrier operates by a uniport-exchange mechanism and provide significant values for the kinetic constants and the equilibrium constants implied in the process. Their analysis leads to the conclusion that the carrier is maximally efficient in the exchange between external malate and internal oxoglutarate, as required by the malate/aspartate shuttle, which should be the main role of the oxoglutarate carrier in brain mitochondria.

Influence of all-trans-retinoic acid on oxoglutarate carrier via retinoylation reaction.

All-trans-retinoic acid (atRA), an activated metabolite of vitamin A, is incorporated covalently into proteins both invivo and invitro. AtRA reduced the transport activity of the oxoglutarate carrier (OGC) isolated from testes mitochondria to 58% of control via retinoylation reaction. Labeling of testes mitochondrial proteins with (3)HatRA demonstrated the binding of atRA to a 31.5 KDa protein. This protein was identified as OGC due to the competition for the labeling reaction with 2-oxoglutarate, the specific OGC substrate. The role of retinoylated proteins is currently being explored and here we have the first evidence that retinoic acids bind directly to OGC and inhibit its activity in rat testes mitochondria via retinoylation reaction. This study indicates the evidence of a specific interaction between atRA and OGC and establishes a novel mechanism for atRA action, which could influence the physiological biosynthesis of testosterone in situations such as retinoic acid treatment.

Synthesized esters of ferulic acid induce release of cytochrome c from rat testes mitochondria.

Ferulic acid plays a chemopreventive role in cancer by inducing tumor cells apoptosis. As mitochondria play a key role in the induction of apoptosis in many cells types, here we investigate the mitochondrial permeability transition (MPT) and the release of cytochrome c induced by ferulic acid and its esters in rat testes mitochondria, in TM-3 and MLTC-1 cells. While ferulic acid, but not its esters, induced MPT and cytochrome c release in rat testes isolated mitochondria, in TM-3 cells we found that both ferulic acid and its esters induced cytochrome c release from mitochondria in a dose-dependent manner, suggesting a potential target of these compounds in the induction of cell apoptosis. The apoptosis induced by ferulic acid is therefore associated with the mitochondrial pathway involving cytochrome c release and caspase-3 activation.

Accelerated photostability study of tretinoin and isotretinoin in liposome formulations.

The photodegradation of retinoic acids, tretinoin and isotretinoin, in ethanol and liposomes was studied. The light irradiation was performed according to the conditions suggested by the ICH Guideline for photostability testing by using a Xenon lamp within a wavelength range of 300-800 nm. The photodegradation process was monitored by UV spectrophotometry. In ethanol solution, tretinoin and isotretinoin undergo complete isomerization just within a few seconds of light exposure to give 13-cis and 9-cis isomers, respectively. The 13-cis isomer from tretinoin undergoes in turn a slow isomerization to the same 9-cis isomer. Both retinoic acids incorporated in liposome complexes showed an increased stability in comparison to the ethanol solutions. In particular for tretinoin, a residual concentration of 60% was still present after a light irradiance of 3470 kJ/m(2), by means of a 250 W/m(2) light power for 240 min, versus a residual value of just 8% measured at the same time in ethanol solution. Moreover, the isomerization rate in liposomes resulted reduced for isotretinoin and practically irrelevant for tretinoin. The degradation rate was found to be dependent on the drug concentration. The better stability of the tretinoin in liposome complex was supposed to be related to its higher incorporation value due to the linear structure of the molecule.

Design and monitoring of photostability systems for amlodipine dosage forms.

Photostability of amlodipine (AML) has been monitored in several pharmaceutical inclusion systems characterized by plurimolecular aggregation of the drug and excipients with high molecular weight. Several formulations including cyclodextrins, liposomes and microspheres have been prepared and characterized. The photodegradation process has been monitored according to the conditions suggested by the ICH Guideline for photostability testing, by using a light cabinet equipped with a Xenon lamp and monitored by spectrophotometry. The formulations herein tested have been found to be able to considerably increase drug stability, when compared with usual pharmaceutical forms. The residual concentration detected in the inclusion complexes with cyclodextrins and liposomes was 90 and 77%, respectively, while a very good value of 97% was found for microspheres, after a radiant exposure of 11,340 kJm(-2).

Retinoylation of proteins in cell-free fractions of rat tissues in vitro.

all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins both in vivo and in vitro. The relative rates of incorporation of all-trans-11,12-(3)H-retinoic acid into rat tissue homogenates in the presence of ATP and coenzyme A were testes>>lung> or =brain> or =kidney>liver. Although all studied cellular organelles of the testes incorporated (3)H-retinoic acid into protein, mitochondria were by far the most active; indeed, up to 25% of the added tritiated retinoic acid (RA) became covalently bound to protein in a 90 min incubation period. In the absence of ATP, coenzyme A, or both cofactors, the amount of RA incorporated into the proteins of testes mitochondria fell to 37%, 16%, and 11%, respectively, of that incorporated in their presence. N-Ethylmaleimide (5 mM) strongly inhibited the reaction. Boiled mitochondria were inactive. After extensive extraction with CHCl(3)-CH(3)OH, the protein-bound radioactivity, which proved largely to be retinoic acid, was released by treatment with proteinase K, hydroxylamine, and dilute base. Thus, retinoic acid is most probably linked to protein as a thiol ester. By SDS-polyacrylamide gel electrophoresis, four protein fractions with molecular masses of approx. 20, 24, 29, and 45 kDa, as well as smaller amounts of larger entities, were labeled in testes mitochondria. The possible identities and roles of these retinoylated proteins are currently being explored.

Synthesis and structure-affinity relationships at the central benzodiazepine receptor of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines.

A series of 2-aryl-3-chloro-2H-pyridazino[4,3-b]indoles, 2-aryl-3-methoxy-2H-pyridazino[4,3-b]indoles, and 2-aryl-2,5-dihydroindeno[1,2-c]pyridazino-3(3H)-ones has been prepared and tested for their ability to inhibit the [3H]flunitrazepam binding to the central benzodiazepine receptor. SAR are presented and discussed in comparison with existing pharmacophore models.

Purification and characterization of the reconstitutively active citrate carrier from maize mitochondria.

The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5'-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min-1) or citrate/malate exchange (0.075 min-1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C.

Changes of mitochondrial properties in maize seedlings associated with selection for germination at low temperature. Fatty acid composition, cytochrome c oxidase, and adenine nucleotide translocase activities

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5 degreesC. When seedlings of the two populations were grown at 14 degreesC (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome c oxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.

Synthesis and binding affinity of 2-phenylimidazo[1,2-alpha]pyridine derivatives for both central and peripheral benzodiazepine receptors. A new series of high-affinity and selective ligands for the peripheral type.

A number of 6-substituted or 6,8-disubstituted alkyl 2-phenylimidazo[1,2-alpha]pyridine-3-carboxylates 5a-h, -acetates 5i-s, 6a-g, and -propionates 5t, 6h and of N,N-dialkyl-2-phenylimidazo[1,2-alpha]pyridine-3-carboxamides 7a-d,-acetamides 7e-t or -propionamide 7u were prepared following new synthetic methods, and their affinities for both the central (CBR) and the peripheral (PBR) benzodiazepine receptors evaluated. The compounds of the ester series displayed low affinity for both receptor types. Conversely, most of N,N-dialkyl(2-phenylimidazo[1,2-alpha]pyridin-3-yl)acetamides 7e-t proved to possess high affinity and selectivity for CBR or PBR depending on the nature of substituents at C(6)- and/or C(8) on the heterocyclic ring system. In particular, the 6-substituted compounds 7f-n displayed ratios of IC50 values (IC50(CBR)/IC50(PBR)) ranging from 0.32 (7m) to 232 (7k), while the 6,8-disubstituted compounds 7o-t were more than 1000-fold more selective for PBR versus CBR. Compounds 7f,m were examined in several different benzodiazepine receptor subtypes. Expression of specific GABAA, receptor subunit assemblies in Xenopus oocytes was utilized to evaluate functionally both the efficacy and potency of the positive modulation of GABA-evoked Cl- currents by 7f and 7m in comparison with Zolpidem. The rank order of potencies of these drugs was 7f (EC50 = 3.2 x 10(-8) M) > Zolpidem (EC50 = 3.6 x 10(-8) M) > 7m (EC50 = 2.2 x 10(-7) M). The actions of these compounds were also tested on alpha 2 beta 2 gamma 2s, receptors. However, the EC50 of these compounds was increased, compared to alpha 1 beta 2 gamma 2s receptors, by 30-, 4-, and 5-fold for 7m, 7f, and Zolpidem, respectively. Finally, these compounds were almost completely devoid of activity at receptors containing the alpha 5 subunit.

Structure-activity relationships of 2-aryl-2,5-dihydropyridazino [4,3-b]indol-3(3H)-ones at the benzodiazepine receptor.

A large series of 2-aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)ones (PIs) carrying properly selected substituents at the indole and N2-phenyl rings was prepared and tested as central benzodiazepine receptor (BZR) ligands and potential (anti)convulsant agents. Stereoelectronic requirements for high receptor affinity were detected by means of 2-D and 3-D QSAR analyses. BZR affinities and pharmacological profiles of the compounds were examined in comparison with some other pyridazinoindolones recently described by us and with pyrazoloquinoline (PQ) analogues. An anticonvulsant activity greater than PQs was generally observed for PIs. Notably, in the test of audiogenically induced seizures, one compound showed a potency comparable to that of diazepam.

Purification and characterization of the reconstitutively active adenine nucleotide carrier from maize mitochondria.

The adenine nucleotide carrier from maize (Zea mays L. cv B 73) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. Sodium dodecyl sulfate-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 32 kD. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 168-fold with a recovery of 60% and a protein yield of 0.25% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ADP, ATP, GDP, and GTP, and was inhibited by atractyloside, bongkrekate, phenylisothiocianate, pyridoxal 5'-phosphate, and mersalyl (but not N-ethylmaleimide). Maximum initial velocity of the reconstituted ATP/ATP exchange was determined to be 2.2 mumol min-1 mg-1 protein at 25 degrees C. The half-saturation constants and the corresponding inhibition constants were 17 microM for ATP, 26 microM for ADP, 59 microM for GTP, and 125 microM for GDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between 0 and 15 degrees C, and 22 kilojoule/mol between 15 and 35 degrees C. Partial amino acid sequences showed that the purified protein was the product of the ANT-G1 gene sequenced previously (B. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303-310).

The role of sterols in the functional reconstitution of water-soluble mitochondrial porins from plants.

Water-soluble porins were prepared from native mitochondrial porins isolated from different plants (pea and corn). In the water-soluble form the porins have lost their channel-forming properties. The water-soluble porins were investigated for the influence of different sterols on their membrane activity and their channel-forming properties in lipid bilayer membranes. Our experiments demonstrated that the water-soluble porins regained channel forming activity when the protein was preincubated with different sterols in the presence of a detergent. The channels formed in lipid bilayer membranes after this procedure regain in many but not all cases the original properties of the native mitochondrial porins. Preincubation with other sterols led to a change in the single-channel conductance or to a complete loss of the voltage dependence. The sterols had also a strong influence on the channel-forming activity of the porins. Preincubation of water-soluble pea porin with the plant sterol beta-sitosterol resulted in a considerable higher channel-forming activity than with all the other sterols used for preincubation. The role of the sterols in the channel-forming complex is discussed.

Formation of beta-glucuronides and of beta-galacturonides of various retinoids catalyzed by induced and noninduced microsomal UDP-glucuronosyltransferases of rat liver.

The complex of microsomal UDP-glucuronosyl transferases (UGT; EC 2.4.1.17) of rat liver catalyzes the formation of retinoyl beta-glucuronide (RAG) from retinoic acid (RA) and retinyl beta-glucuronide (ROG) from retinol (ROL) in the presence of UDP-glucuronic acid (UDPGA). The relative rates of formation of beta-glucuronides in noninduced microsomes were: 9-cis RA > 13-cis RA > all-trans 4-oxo RA > TTNPB > all-trans RA > CD-367 > 13-cis ROL > 9-cis ROL > acitretin > all-trans ROL. Michaelis constants (KM) for all-trans RA, 13-cis ROL, TTNPB and UDPGA were 130 microM, 300 microM, 210 microM and 2.6 microM, respectively. Galacturonides of RA, but not detectably of ROL, were formed from UDP-galacturonic acid at 11-30% the rate of the beta-glucuronides, whereas UDP derivatives of nonionized sugars did not serve as substrates. Pretreatment of rats with 3-methylcholanthrene markedly increased RAG formation in the absence of detergent (Triton X-100), but less so in its presence. Clofibrate pretreatment doubled the rate of RAG formation, whereas phenobarbital pretreatment showed little effect. N-Ethylmaleimide (5 microM) minimized cis-trans isomerization without significantly affecting glucuronidation. Rates of glucuronidation of retinoids clearly depend both on their isomeric states and on their chemical structures. Different UGT enzymes might well act on different geometric isomers of RA.

Synthesis, benzodiazepine receptor affinity and anticonvulsant activity of 5-H-indeno[1,2-c]pyridazine derivatives.

Title compounds 1-36 were synthesized by reacting hydrazine with aldol adducts obtained from ninhydrin and methyl or alpha methylene ketones and aldehydes. Some of them showed a low, but significant benzodiazepine receptor affinity whose variation was interpreted through a structure-activity relationships study based on qualitative correlations and Comparative Molecular Field Analysis (CoMFA). Some indeno-pyridazine derivatives were found to possess an interesting anticonvulsant activity which however does not seem simply related to the benzodiazepine receptor modulation.

2-Aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)-ones: novel rigid planar benzodiazepine receptor ligands.

A series of 2-aryl-2,5-dihydropyridazino[4,3-b]indol-3(3H)-ones 5 were prepared and evaluated for their ability to inhibit radioligand binding to BZR, and to prevent sound and pentylenetetrazole (PTZ) induced seizures in mice. The biological and pharmacological results are discussed in the light of some recently proposed pharmacophore models and compared through molecular orbital and molecular modeling studies to those obtained from the close pyrazoloquinoline analogs 6.

Pyrrolo[1',2':1,2]imidazo[4,5-b]pyridines, pyrrolo- [2',1':2,3]imidazo[4,5-c]pyridines and pyrrolo[2,1-f]purines as potential benzodiazepine ligands.

The synthesis of some 7,8,8a,9-tetrahydro-6H-pyrrolo[1',2':1,2]imidazo[4,5-b]pyridin-6-ones, 5,5a,6,7-tetrahydro-8H-pyrrolo[2',1':2,3]imidazo[4,5-c]pyridin-8-ones and 7,8,8a,9-tetrahydro-6H-pyrrolo[2,1-f]purin-6-ones is reported. The structure of the obtained compounds has been assigned by means of 1H-NMR spectra assisted by NOESY measurements. In addition, the ability to displace [3H]-flunitrazepam binding from rat brain membranes is determined. Only the pyrrolopurine derivative 5d binds to the benzodiazepine receptor (BZR) with appreciable potency.

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria.

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.

Partial Purification and Reconstitution of the alpha-Ketoglutarate Carrier from Corn (Zea mays L.) Mitochondria.

The alpha-ketoglutarate carrier from corn shoot mitochondria (Zea mays L., B 73) was solubilized in Triton X-114 and partially purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. On SDS-gel electrophoresis, the hydroxyapatite/celite eluate showed various protein bands between 12 and 70 kilodaltons. When reconstituted into liposomes, the alpha-ketoglutarate transport protein catalyzed a phthalonate-sensitive alpha-ketoglutarate/alpha-ketoglutarate exchange. The protein was purified 60-fold with a recovery of 88% with respect to the mitochondrial extract. The protein yield was 0.6%. The properties of the reconstituted carrier, i.e. requirement for a counter-anion, substrate specificity, and inhibitor sensitivity, were similar to those of the alpha-ketoglutarate transport system as characterized in plant and animal mitochondria.