Identification of slow motions in the reduced recombinant high-potential iron sulfur protein I (HiPIP I) from Ectothiorhodospira halophila via 15N rotating-frame NMR relaxation measurements

The 2-methyl branched-chain enoyl CoA reductase (ECR) plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode, Ascaris suum. Two-dimensional gel electrophoresis of the purified ECR yielded multiple spots, with two distinct but overlapping N-terminal sequences. These multiple isoforms were not the result of population effects, as the pattern observed on 2-D gels of the purified ECR was identical to those on immunoblots of muscle homogenates isolated from individual worms. A full-length cDNA coding for the major ECR isoform (ECRI) has been cloned and sequenced and compared with that of the minor isoform (ECRII) which has been described previously (Duran et al. J Biol Chem 1993;268:22391-22396). ECRI contained the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a 5' untranslated region (UTR) of 13 nucleotides, an open reading frame (ORF) of 1257 nucleotides, a 3'-UTR of 110 nucleotides that included the polyadenylation signal AATAAA downstream of the termination codon and a short poly(A) tail. The ORF predicted a 16 amino acid leader sequence not found in the native protein and a mature protein of 403 amino acids with a molecular weight of 43 698 and a predicted pI of 6.2. ECRI and ECRII were 73% identical at the predicted amino acid level and their mRNAs exhibited significant structural similarity even though they were products of separate genes. Comparison of ECRI and ECRII with the sequences of acyl CoA dehydrogenases from a variety of different sources revealed a high degree of interspecies sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene. This result is surprising since the ascarid enzymes function as reductases, not as dehydrogenases. Both ECRs were tissue-specific and developmentally regulated and were found in transitional third-stage larvae (L3) and adult muscle, but not in early, aerobic larval stages or adult testis, ovary, or intestine. The ratio of ECRII to ECRI was greater in L3 than in adult muscle. Interestingly, both ECRs also appeared to be expressed in pharyngeal muscle, suggesting that branched-chain fatty acid synthesis may not be confined exclusively to body wall muscle.     

Wild isolates of murine cytomegalovirus induce myocarditis and antibodies that cross-react with virus and cardiac myosin

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic metabolism of the parasitic nematode Ascaris suum. Two isoforms of the alpha-subunit of pyruvate dehydrogenase (E1) have been identified: alpha I is most abundant in anaerobic adult muscle and alpha II in aerobic larvae. Both isoforms have been expressed as alpha 2 beta 2 tetramers with a muscle-specific beta-subunit, purified to apparent homogeneity, reconstituted with E1-deficient adult A. suum muscle PDC, and assayed for PDC and E1 kinase activity. Recombinant alpha II is a poor substrate for the adult E1 kinase, but its stoichiometry of phosphorylation/inactivation is similar to that reported for the human E1. Initially, inactivation parallels the incorporation of about 1 mol 32P/mol E1 and at maximal phosphorylation about 2.4 32P/mol E1 is incorporated. In contrast, recombinant alpha I (r alpha I) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivation. To examine this altered pattern of phosphorylation, the two phosphorylation sites in each E1 alpha subunit of the r alpha I (site 1 and site 2) were changed either individually or together from Ser to Ala by site-directed mutagenesis. Site 1 was phosphorylated more rapidly than site 2, but the phosphorylation of either site resulted in inactivation, and the phosphorylation of only a single E1 alpha subunit of the tetramer was necessary for inactivation. However, both E1 alpha subunits of the tetramer were phosphorylated, based on the incorporation of about 3.5 mol 32P/mol E1 at maximal phosphorylation and the altered mobility of most of the E1 alpha subunits during SDS-PAGE. These observations suggest that the regulation of both E1 isoforms is modified to maintain PDC activity during the transition to anaerobiosis.     

Identification of a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum

A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino-terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45-enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD+ ratios associated with anaerobiosis.     

Cloning, expression, and properties of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase

cDNA encoding the regulatory subunit of bovine mitochondrial pyruvate dehydrogenase phosphatase (PDPr) has been cloned. Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA. The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculated Mr of 95,656. This value is in agreement with the molecular mass of native PDPr (95,800 +/- 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry. The mature form of PDPr was expressed in Escherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity. It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine. A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine dehydrogenase, which functions in choline degradation.     

Detection of a homozygous four base pair deletion in the protein X gene in a case of pyruvate dehydrogenase complex deficiency

While the presence of a lipoyl-containing protein (protein X) separate from lipoyl transacetylase in the pyruvate dehydrogenase complex (PDC) has been known for some time, until recently only the cDNA for the yeast enzyme has been cloned. We have cloned, sequenced and characterized the cDNA encoding the human protein X and localized the protein X gene to chromosome 11p13. We also report here a new case of protein X deficiency identified immunologically, with decreased activity of PDC and without mutations in the E1alpha subunit or E1beta subunit. We report that the cDNA and gene of this patient for protein X has a homozygous 4 bp deletion, specifically in the putative mitochondrial targeting signal sequence which results in a premature stop codon. This is the first documented case of a molecular defect in pyruvate dehydrogenase protein X.     

Application of anti-E1 monoclonal antibodies to the study of the pyruvate dehydrogenase complex

It has been shown that monoclonal antibody (mAb) F7F10 raised against pyruvate dehydrogenase component (E1) of pigeon breast muscle pyruvate dehydrogenase complex (PDC) has no influence on the E1 activity, measured in the system with artificial oxidants. However it inhibited the full NAD+ and coenzyme A dependent activity of PDC. The competition of the F7F10 antibody with the E2 component of PDC for the binding with E1 was revealed by immunoenzymatic and kinetic analysis. It is suggested that F7F10 mAb interacts with an antigenic determinant, located in the immediate vicinity of or overlapping with the E1 region, responsible for the interaction with the E2 component of PDC.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme

The nucleotide sequence of a full-length cDNA encoding NAD(+)-malic enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2269 bases comprises a 5'-leader, a single open reading frame of 1851 bases, and the complete 3'-noncoding region of 340 bases. The first 12 amino acids of the translated sequence are hydrophobic, typical of mitochondrial translocation signals, and do not appear in the purified mature protein. The mature protein contains 605 amino acids and has a molecular mass of 68,478 Da. The amino acid sequences of tryptic peptides from the purified protein and also the N-terminal sequence show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the ascarid protein with the human and rat liver NAD(+)-malic enzymes reveals highly conserved regions interrupted with long stretches of lesser homologous sequences. Structural motifs such as the putative nucleotide binding domains and also the malate binding site are clearly identified by alignment of the three protein sequences.     

Isolation, pharmacology and gene organization of KPSFVRFamide: a neuropeptide from Caenorhabditis elegans

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH]+) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, >/=0.1 microM) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, >/=10 nM).     

Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cDNA library

A full-length cDNA clone for human 6-phosphogluconate dehydrogenase (PGD) was isolated from a human adult heart cDNA library. The clone encoded an open reading frame of 483 amino acids. When the amino acid sequences of human PGD and sheep PGD were aligned, 94.2% identity between these two proteins was found. Its calculated molecular weight is 53,149 daltons. The predicted isoelectric point is 6.85. When the secondary structure of human PGD was examined by the PROSIS software, 36% alpha-helix and 9% beta-sheet were found.     

Mutations in PDX1, the human lipoyl-containing component X of the pyruvate dehydrogenase-complex gene on chromosome 11p1, in congenital lactic acidosis

We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression.     

Molecular cloning and expression of rabbit pancreatic cholesterol esterase

Rabbit pancreatic cholesterol esterase (CEase, carboxyl ester lipase, EC 3.1.1.3) has been cloned from a lambda gt11 library of adult rabbit pancreatic cDNA. The open reading frame consists of 1788 nucleotides which encodes 576 amino acids of the functional protein and a 20 amino acid leader peptide. When compared to other species, the greatest homology is observed between residues 82-248 with little or no homology at the C-terminal end where proline-glutamate-serine-threonine (PEST) segments are a characteristic feature of the human CEase. Rabbit CEase (RCEase) retains the active-site serine (gxsxg), the active-site histidine and the tentative heparin binding site (KKRCLQ) at similar positions in comparison to pancreatic CEases of other species. When rabbit CEase cDNA is expressed in monkey kidney (COS-7) cells, enzymatic hydrolytic activity is detected in the growth medium as is a 67 kDa protein by Western blotting with polyclonal anti-CEase antibody. Northern blot analysis shows two mRNA (2.2 and 3.2 kb) species.     

Rationale for a trial of prevention of perinatal transmission of hepatitis C via specific immunoglobulins

Insects perceive a large number of airborne chemicals as olfactory components mainly through the antenna. It is thought that detection of the odorants by specific receptors is followed by a degradative pathway that clears the olfactory organ from accumulating chemicals. In Drosophila, a number of P450 monooxygenases are involved in the metabolism of foreign chemicals [Dunkov et al. (1996) Cytochrome P450 gene cluster in Drosophila melanogaster, Mol. Gen. Genet. 251, 290-297]. NADPH-cytochrome P450 oxidoreductases serve to transfer reducing equivalents to P450 monooxygenases. We isolated cDNA and genomic clones coding for a Drosophila NADPH cytochrome P450 oxidoreductase (CPR). The largest cDNA of 2471 nucleotides in length contained an open reading frame of 693 amino acids that includes the putative CPR sequence. CPR is a single copy gene as shown by genomic Southern hybridisation and maps to the cytogenetic map position 26C on the second chromosome. Comparison of genomic and cDNA CPR sequences revealed a gene structure that is split into at least six exons. The CPR protein sequence is almost identical with that of house fly and remarkably conserved when compared to vertebrates and yeast. RNA expression is high in embryos and antennae as compared to adult heads, adult bodies and larvae. High expression in antennae may reflect the putative function in olfactory clearance.     

Deficiency of dihydrolipoamide dehydrogenase due to two mutant alleles (E340K and G101del). Analysis of a family and prenatal testing

A male child with metabolic acidosis was diagnosed as having dihydrolipoamide dehydrogenase (E3) deficiency. E3 activity of the proband's cultured fibroblasts and blood lymphocytes was 3-9% of normal, while in the parent's lymphocytes it was about 60% of normal. The proband's pyruvate dehydrogenase complex (PDC) and the alpha-ketoglutarate dehydrogenase complex activities from cultured skin fibroblasts were 12% and 6% of normal, respectively. PDC activity in the parents cultured fibroblasts was 25-31% of normal. Western and Northern blot analyses showed similar quantities of E3 protein and mRNA in cultured fibroblasts from the proband and his parents. DNA sequencing of cloned full-length E3 cDNAs, from the proband and the parents, showed two mutations on different alleles of proband were inherited from the parents. One mutation is a three nucleotide (AGG) deletion, from the mother, resulting in deletion of Gly101 in the FAD binding domain. The other mutation is a nucleotide substitution (G to A), from the father, leading to substitution of Lys for Glu340 in the central domain. The same deletion mutation was found in E3 cDNA from a chorionic villus sample and cultured fibroblasts obtained from the mother's subsequent offspring. This finding illustrates the possibility of successful prenatal diagnosis of E3 deficiency utilizing mutations characterized prior to initiation of pregnancy.