AT1 receptor blockade alters metabolic, functional and structural proteins after reperfused myocardial infarction: detection using proteomics

Angiotensin II (AngII) type 1 receptor (AT1R) blockers (ARBs) limit left ventricular (LV) dysfunction and necrosis after reperfused myocardial infarction (RMI) and proteomics can detect changes in protein levels after injury. We applied proteomics to detect changes in levels of specific protein in the ischemic zone (IZ) and non-ischemic zone (NIZ) of dog hearts after in vivo RMI (90 min of anterior ischemia; 120 min of reperfusion) and treatment with intravenous vehicle (control) and the ARBs valsartan or irbesartan (10 mg/kg) over 30 min before RMI. We also assessed LV function, infarction and apoptosis. Both ARBs limited the RMI-induced LV dysfunction, infarct size and apoptosis. Proteomics detected differential expression of 5 randomly selected proteins in the IZ compared to the NIZ after RMI: decrease in a subunit of ATP synthase isoform precursor (consistent with increased conversion to a subunit under metabolic stress), M chain creatine kinase (consistent with cellular damage) and ventricular myosin light chain-1 (consistent with structural damage and decreased contractility); and increase in NAD+ -isocitrate dehydrogenase (ICDH) and alpha subunit and ATP synthase D chain (mitochondrial, consistent with metabolic dysfunction). Importantly, changes in NAD+ -ICDH and ATP synthase D chain were reversed by ARB therapy. Thus, proteomics can detect regional changes in metabolic, contractile, and structural proteins after RMI and several of these proteins are favorably modified by ARBs, suggesting that they may be novel therapeutic targets.     

Detection of regional changes in protein levels in the in vivo canine model of acute heart failure following ischemia-reperfusion injury: functional proteomics studies

In this study, we demonstrate the use of proteomics to detect regional differences in protein levels between the reperfused ischemic zone (IZ) and the nonischemic zone (NIZ) of dog hearts which were subjected to in vivo ischemia-reperfusion injury. Using the two-dimensional gel electrophoresis (2-DE) technique, we identified five proteins that were differentially expressed in the IZ versus NIZ: (1) the alpha subunit of ATP synthase isoform precursor was decreased 1.71-fold; (2) creatine kinase M chain was decreased 1.72-fold; (3) NAD+-isocitrate dehydrogenase, alpha subunit was increased 8.34-fold; (4) ATP synthase D chain, mitochondrial was increased 3.02-fold; (5) ventricular myosin light chain-1 was decreased 2.02-fold. Additionally, we found that the level of actin was decreased 2.6-times in the IZ compared to the NIZ on Western blot analysis but was unchanged on 2-DE.     

Valsartan reverses post-translational modifications of the delta-subunit of ATP synthase during in vivo canine reperfused myocardial infarction

To determine whether reperfused myocardial infarction (RMI) induces PTM of the delta-subunit of the mitochondrial metabolic enzyme ATP synthase (ATP/delta) in the ischemic zone (IZ) and whether this can be reversed by the angiotensin II type 1 receptor (AT(1)R) blocker valsartan, we applied a pharmaco-proteomics approach in canine RMI hearts with or without valsartan pretreatment. Using the 2-DE technique, we identified differential regional expression of ATP/delta in the IZ compared to the non-ischemic zone (NIZ), with an approximately 2-fold increase in the IZ that was normalized by valsartan. Furthermore in the IZ, RMI triggered S-nitrosylation of cysteine-100, nitration of the two tyrosines 88 and 225, and hydroxylation of lysine-182 in ATP/delta followed by its myristoylation. Importantly, valsartan abolished these modifications of ATP/delta in the IZ, triggered phosphorylation of serine-76 in both the IZ and NIZ, and decreased necrosis, apoptosis, left ventricular dysfunction and remodeling. Thus, AT(1)R-blocker-induced cardioprotection during RMI is associated with phosphorylation of ATP/delta and inhibition of nitric oxide-related chemical modifications such as S-nitrosylation, nitration and hydroxylation. Targeting specific PTMs during RMI, such as those of ATP/delta with AT(1)R blockade, might be a potentially powerful novel therapeutic approach. However, the identification of S-nitrosylation was putative and requires MS/MS verification.     

Rotation of the γ Subunit in F1-ATPase; Evidence That ATP Synthase Is a Rotary Motor Enzyme

ATP-dependent, azide-sensitive rotation of the subunit relative to the ₃₃ hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.     

Chromosomal localization of genes required for the terminal steps of oxidative metabolism: α and γ subunits of ATP synthase and the phosphate carrier

The terminal steps of oxidative phosphorylation include transport of phosphate and ADP into the mitochondrial matrix, synthesis of ATP in the matrix, and transport of the product ATP into the cytosol where it can be utilized to perform cellular work. Three nuclear genome encoded membrane proteins, namely, the phosphate carrier (PHC), the adenine nucleotide carrier (ANT), and the ATP synthase complex, consisting of at least 13 individual subunits, catalyze these reactions. The locations of the and subunits of the mitochondrial ATP synthase complex and the mitochondrial phosphate carrier, PHC, on human chromosomes were determined using cloned rat liver cDNA as probes. Human homologues of the subunit are on chromosomes 9 and 18, the subunit are on chromosomes 10 and 14, and the PHC was localized to chromosome 12.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

Genes encoding the H,K-ATPase α and Na,K-ATPase α3 subunits are linked on mouse Chromosome 7 and human Chromosome 19

We have used linkage analysis and fluorescence in situ hybridization to determine the chromosomal organization and location of the mouse (Atp4a) and human (ATP4A) genes encoding the H,K-ATPase subunit. Linkage analysis in recombinant inbred (BXD) strains of mice localized Atp4a to mouse Chromosome (Chr) 7. Segregation of restriction fragment length polymorphisms in backcross progeny of Mus musculusxMus spretus mating confirmed this assignment and indicates that Atp4a and Atp1a3 (gene encoding the murine Na,K-ATPase 3 subunit) are linked and separated by a distance of 2 cM. Analysis of the segregation of simple sequence repeats suggested the gene order centromere-D7Mit21-D7Mit57/Atpla3-D7Mit72/Atp4a. A human Chr 19-enriched cosmid library was screened with both H,K-ATPase and Na,K-ATPase 3 subunit cDNA probes to isolate the corresponding human genes (ATP4A and ATP1A3, respectively). Fluorescence in situ hybridization with gene-specific cosmid clones localized ATP4A to the q13.1 region, and proximal to ATP1A3, which maps to the q13.2 region, of Chr 19. These results indicate that ATP4A and ATP1A3 are linked in both the mouse and human genomes.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

α-Isopropylmalate synthase as a marker for the leucine biosynthetic pathway in several clostridia and in Bacteroides fragilis

-Isopropylmalate synthase (EC 4.1.3.12) is present in extracts of Bacteroides fragilis, Clostridium thermoaceticum, Clostridium formicoacetium, Clostridium pasteurianum, and Clostridium kluyveri with specific activities (mol -isopropylmalate formed per min and g protein) of 8.6, 8.9, 2.4, 1.9, and 0.3, respectively. The product -isopropylmalate was identified by gas chromatography combined with mass spectroscopy. The presence of 5 mM leucine in the growth medium represses the synthesis of -isopropylmalate synthase in C. thermoaceticum by 40 and 70 %. The enzyme from C. pasteurianum was partially purified to a specific activity of 1413. All studied enzyme properties are similar to those of the enzymes from aerobic bacteria. It is suggested that in these anaerobic bacteria the -isopropylmalate pathway is present in addition to the pathway via the ferrodoxin-dependent, reductive carboxylation of branched chain fatty acids.Abbreviations used -KIV -Ketoisovalerate - -IPM -Isopropylmalate - CoA Coenzyme A     

Angiotensin receptor blockade and angiotensin-converting-enzyme inhibition limit adverse remodeling of infarct zone collagens and global diastolic dysfunction during healing after reperfused ST-elevation myocardial infarction

To determine whether therapy with the angiotensin II type 1 receptor blocker (ARB) candesartan and the comparator angiotensin-converting-enzyme inhibitor (ACEI) enalapril during healing after reperfused ST-elevation myocardial infarction (RSTEMI) limit adverse remodeling of infarct zone (IZ) collagens and left ventricular (LV) diastolic dysfunction, we randomized 24 dogs surviving anterior RSTEMI (90-min coronary occlusion) to placebo, candesartan, and enalapril therapy between day 2 and 42. Six other dogs were sham. We measured regional IZ and non-infarct zone (NIZ) collagens (hydroxyproline; types I/III; cross-linking), transforming growth factor-β (TGF-β) and topography at 6 weeks, and hemodynamics, LV diastolic and systolic function, and remodeling over 6 weeks. Compared to sham, placebo-RSTEMI differentially altered regional collagens, with more pronounced increase in TGF-β, hydroxyproline, and type I, insoluble, and cross-linked collagens in the IZ than NIZ, and increased IZ soluble and type III collagens at 6 weeks, and induced persistent LV filling pressure elevation, diastolic and systolic dysfunction, and LV remodeling over 6 weeks. Compared to placebo-RSTEMI, candesartan and enalapril limited adverse regional collagen remodeling, with normalization of type III, soluble and insoluble collagens and decrease in pyridinoline cross-linking in the IZ at 6 weeks, and attenuation of LV filling pressure, diastolic dysfunction, and remodeling over 6 weeks. The results suggest that candesartan and enalapril during healing after RSTEMI prevent rather than worsen adverse remodeling of IZ collagens and LV diastolic dysfunction, supporting the clinical use of ARBs and ACEIs during subacute RSTEMI.     

The b Subunit of Escherichia coli ATP Synthase

The b subunit of ATP synthase is a major component of the second stalk connecting the F₁and F₀ sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF₁, where they interact with the subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal -binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the -binding domain is more globular. Sites ofcrosslinking between b and the a, , , and subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.     

The L-type calcium channel alpha 1C subunit gene undergoes extensive,uncoordinated alternative splicing

The _1C subunit is the pore-forming protein for the L-type calcium channel. Previous studies indicate that there is possible tissue-specific alternative splicing of this gene. In this study we cloned the entire open reading frame of the _1C subunit cDNA from adult rat cardiac myocytes in a single piece (6.64 kb). Using 75 positive clones that were identified by restriction enzyme mapping, we tested the alternative splicing patterns of the Ca_v1.2 gene that encodes the _1C subunit protein and focused on five loci: IS6, post-IS6, IIIS2, IVS3, and the c-terminus. The results indicate that: (1) alternative splicing occurs in most of the loci, giving rise to two or three different isoforms at those sites; (2) there is a predominant form for each splicing site, (3) there does not appear to be consistent coordination of splicing at multiple loci of this gene. Alternative splicing is not tissue-specific in most regions. (Mol Cell Biochem 269: 153–163, 2005)     

Efficient membrane targeting of the chick nicotinic acetylcholine receptor α-subunit in stable insect cell lines

We have synthesised the -subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChR, encoding the -subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR -subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - BTX -bungarotoxin - BSA bovine serum albumin - FITC Fluoroscein isothiocyanate - G418 geneticin-418 - hpi hours post-infection - ie-1 immediate early 1 gene - nAChR nicotinic acetylcholine receptor alpha subunit - Sf Spodoptera frugiperda - tPA tissue plasminogen activator     

DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

The highly purified DNA Pol- from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase- showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231–6237].The catalytic polypeptide, DNA polymerase- of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase- from embryonic chicken brain (ECB) contains an -galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase- reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567–573] by the treatment with methyl--galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (M _r = 186 kDa) with an -galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA.Pol- as determined by immunostaining with the polymerase--specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA- and a complete separation of polymerase complex and primase.     

Characteristics of antibodies to guinea pig (Na++K+)-adenosine triphosphatase and their use in cell-free synthesis studies

Summary Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the (catalytic) and the (glycoprotein) subunits of Na/K-ATPase. The titers of the anti- and anti- antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti- subunit from anti- subunit antibodies. There was not cross-reactivity of antibodies to one subunit with the other subunit. The subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979,J. Biol. Chem. 254:2221–2228). Anti- antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the subunit of Na/K-ATPase from human, cow, dog, rabbit, rat mouse, turtle, and toad; and with the subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The antisera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized chain was 96,000 daltons. Its identity was established with purified anti- antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the subunit was not detected in this system.     

The effect of modification and fragmentation of α-lactalbumin on lactose and lactosamine synthase reactions

In the presence of the modifier protein -lactalbumin, bovine milk galactosyltransferase transfers galactose to glucose forming lactose instead of transferring toN-acetylglucosamine formingN-acetyllactosamine. At low concentrations of -lact-albumin, the lactosamine synthase activity is stimulated by -lactalbumin and decreases when the lactose synthase activity develops along a sigmoidal curve. The observation suggests that different interactions between -lactalbumin and enzyme were responsible for the modulating effect of the -lactalbumin in the lactose and lactosamine synthase reactions.To study the nature of the protein-protein interactions, -lactalbumin was both modified and cleaved chemically. Reduction and alkylation with iodoacetic acid, iodoacetamide or 4-vinylpyridine abolished the ability of the -lactalbumin to induce lactose synthase activity but stimulated lactosamine synthase activity 7-to 12-fold.A peptide fragment corresponding to residues 26–60 of -lactalbumin isolated from a 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene (BNPS-skatole) fragmentation of the molecule was active in the lactosamine but not lactose synthase reaction. We concluded that, whereas lactose synthase required -lactalbumin, in the native conformation, lactosamine synthase activity was stimulated by a linear sequence of amino acids in peptide 26–60.Abbreviations MES 4-N-morpholinoethanesulfonic acid - TRIS 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP-Gal uridinediphosphogalactose - BNPS-skatole 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene - EDTA ethylene diamine tetra acetic acid     

Extensive variation at the α-glycerophosphate dehydrogenase locus in species of waterstriders (Gerridae: Hemiptera)

Variation at the -glycerophosphate dehydrogenase (-Gpdh; EC 1.1.1.8) locus was surveyed in 11 species of waterstriders (Gerridae: Hemiptera) and five other species of aquatic Hemiptera. Species of waterstriders exhibited considerable inter- and intraspecific variation in degree of winglessness. Average heterozygosity (0.401±0.090) and average number of observed electromorphs (5.36±0.96) for the 11 gerrid species were well above values reported for nearly all other insect species surveyed to date. Wing-monomorphic and wing-polymorphic species did not differ in average -Gpdh heterozygosity. Of the three wing-polymorphic species surveyed geographically, two species exhibited marked variation in wing-morph frequencies but homogeneous -Gpdh allele frequencies. The third species exhibited geographically homogeneous -Gpdh and wing-morph frequencies, but no significant association between -Gpdh phenotype and wing morph was observed in any surveyed population. These results are consistent with hypotheses evoking either relaxed purifying selection at the -Gpdh locus in species of Gerridae due to the apparent reduced importance of flight, or selective maintenance of common -Gpdh electromorphs.This work was supported by NSF Grant DEB 76-20967 to Alan H. Brush, funds from the Research Foundation of the University of Connecticut to Carl W. Schaefer, and USPHS Grant GM 21133 to Richard K. Koehn.     

Molecular cloning,characterization and expression of an elongation factor 1α gene in maize

A cDNA (zmEF1A) and the corresponding genomic clone (zmgEF1A) of a member of the gene family encoding the subunit of translation elongation factor 1 (EF-1) have been isolated from maize. The deduced amino acid sequence is 447 residues long interrupted by one intron. Southern blot analysis reveals that the cloned EF-1 gene is one member out of a family consisting of at least six genes. As shown by northern hybridizations in leaves the mRNA level increases at low temperature whereas time-course experiments over 24 h at 5°C show that in roots the overall mRNA level of EF-1 is transiently decreased. These results indicate that the expression of EF-1 is differently regulated in leaves and roots under cold stress.