Mitochondrial ATP synthase. Interaction of a synthetic 50-amino acid, beta-subunit peptide with ATP

A 50-amino acid peptide predicted by chemical modification studies of F1 and by comparison with adenylate kinase to comprise part of an ATP-binding domain within the beta-subunit of mitochondrial ATP synthase has been synthesized and purified. In the numbering system used for bovine heart beta, the peptide consists of amino acid residues from aspartate 141 at the N-terminal end to threonine 190 at the carboxyl end. In Tris-Cl buffer, pH 7.4, the peptide undergoes a dramatic reaction with ATP resulting in precipitate formation. Analysis of the precipitate shows it to contain both peptide and ATP. Similar to the ATPase activity of F1 and the binding of nucleotide to the enzyme, the capacity of ATP to induce precipitation of the peptide is decreased markedly by lowering pH. Interaction of the peptide with the fluorescent ATP analog, TNP-ATP (2'(3')-O-(2,4-6-trinitrophenyl)-adenosine 5'-triphosphate), can be demonstrated in solution at low concentrations. A 7-fold enhancement in fluorescence is observed when 2.5 microM TNP-ATP interacts with 2.5 microM peptide. Divalent cation is neither required for ATP-induced precipitation of the peptide nor for demonstrating interaction between TNP-ATP and peptide, just as Mg2+ is not required for nucleotide binding to F1. These results indicate that the beta-subunit peptide studied here comprises at least part of a nucleotide-binding domain within the mitochondrial ATP synthase complex.     

ATP synthases: insights into their motor functions from sequence and structural analyses

ATP synthases are motor complexes comprised of F₀ and F₁ parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F₁. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F₁ to the rotor of F₀. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits.     

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.     

ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity.

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

Elements in autopsy liver tissue samples from Greenlandic inuit and Danes. V. Selenium measured by X-ray fluorescence spectrometry

The content of selenium in normal liver tissue samples from Greenlandic Inuit was measured and the results compared with those obtained in normal liver tissue samples from Danes. Normal liver tissue samples were obtained at autopsy from 50 Greenlandic Inuit (27 men, 23 women) with a median age of 61 years (range 23–83) and from 74 Danes (44 men, 30 women) with a median age of 60 years (range 15–87). Total liver selenium content was measured by X-ray fluorescence spectrometry. The content of selenium (median) was in Inuit 26.6 mol/kg dry liver (5–95 percentile: 15.2–49.4) and in Danes 17.7 mol/kg dry liver (5–95 percentile: < 3.8–36.5) (p < 0.0001). Liver selenium content displayed no significant gender difference, either in Inuit or Danes. In Inuit men, there was a negative correlation between liver selenium content and age (r_s = −0.39, p < 0.05), whereas Danish men displayed a positive correlation between liver selenium content and age (r_s = 0.37, p = 0.02). There was no correlation in Inuit or Danish women. In Inuit, the median hepatic selenium index (liver selenium content divided by age) was 0.48 and in Danes 0.33 (p = 0.001). There was an inverse correlation between hepatic selenium index and age both in Inuit (r_s = −0.77, p < 0.0001) and in Danes (r_s = −0.47, p < 0.0001). In conclusion, Inuit had a higher liver content of selenium and a higher hepatic selenium index compared with Danes. The more favourable selenium status is due to a higher nutritional selenium intake with fish and meat from sea mammals.     

Synthesis and incorporation of an alpha-hexofuranosyl thymidine into oligodeoxynucleotides via its two exocyclic OH-groups

1-(2,3-Dideoxy-3-amino-alpha-D-arabino-hexofuranosyl)thymine is considered as a conformationally restricted acyclic nucleoside using the furanose ring to link the diol backbone to the nucleobase. The appropriately substituted phosphoramidites were synthesised via 1-(5,6-di-O-acetyl-2,3-dideoxy-3-phthalimido-alpha-D-arabino-hexofuranosyl)thymine and used in oligodeoxynucleotide (ODN) synthesis. However, the binding affinity of the mixed ODNs towards complementary DNA and RNA was decreased compared to the wild-type oligos. The decrease was smaller when the monomer was inserted near the end of the sequence. The insertions into an alpha T sequence or in a beta T sequence gave nearly the same dropping in melting temperature per modification which indicates that the new nucleotide modifications behave both as alpha and beta nucleotides.