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L J Reed

Publications and source records attributed to L J Reed.

At least 37 records · Page 2Linked to original sources
PubMed1996

Stoichiometry of binding of mature and truncated forms of the dihydrolipoamide dehydrogenase-binding protein to the dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

The dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2). To gain further insight into the number and localization of binding sites for E3BP on the 60-mer E2, truncated forms of the E3BP lacking the lipoyl and E3-binding domains were engineered by deletion mutagenesis. The recombinant proteins contained a polyhistidine extension on the amino terminus to facilitate purification to near-homogeneity. The stoichiometry of binding of the truncation mutants to a truncated form (inner core) of E2 (tE2, residues 181-454), lacking the lipoyl domain and the E1-binding domain, was determined. Mixtures containing tE2 and excess intact or truncated forms of E3BP were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or tE3BP, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that tE2 binds about 20 copies of intact E3BP-H, about 24 copies of tE3BP-H144 (residues 144-380), lacking the lipoyl domain, and about 31 copies of tE3BP-H218 (residues 218-380), lacking both the lipoyl and E3-binding domains. The results indicate that there apparently is a binding site for E3BP on each E2 subunit and that steric hindrance by segments of E3BP prevents full stoichiometric binding of E3BP to the pentagonal dodecahedron-like E2.

Acetyltransferases↗
PubMed1996

Significance of metabolism in the disposition and action of the antidysrhythmic drug, dofetilide. In vitro studies and correlation with in vivo data.

Dofetilide, a class III antidysrhythmic agent, undergoes both renal and metabolic clearance. Characterization of the metabolism in vitro allows explanation of species differences, whereas identification of the human enzymes involved permits assessment of potential drug interaction. In liver microsomes, the rate of oxidative metabolism of dofetilide is in the order: male rat > female rat > dog > humans, which correlates with the metabolic clearance seen in vivo. In vitro products of oxidative metabolism, formed by N-dealkylation, are the same as those formed in vivo, with the N-desmethyl being the major product. This route of dofetilide metabolism is mediated by cytochrome P450 (CYP). In humans, N-demethylation has a high KM of 657 +/- 116 microM, indicating low affinity for the enzyme's active site. In a number of human liver microsomal preparations, this rate correlated (r = 0.903) with the activity of CYP3A4. There was no correlation with the activities of other isozymes. Specific isozyme inhibitors also indicated the involvement of CYP3A4, with partial inhibition being observed with ketoconazole and troleandeomycin, whereas the activator, alpha-naphthaflavone, caused increased turnover. No inhibition was observed with specific inhibitors or competing substrates for other isozymes. Dofetilide did not significantly inhibit CYP2C9, CYP2D6, or CYP3A4 at concentrations up to 100 microM in vitro. In contrast, amiodarone (IC50, 25 microM) and flecainide (49 microM) inhibited CYP2C9 and quinidine (0.26 microM), and flecainide (0.44 microM) inhibited CYP2D6. Many antidysrhythmic drugs have active, circulating metabolites, complicating the relationship of dose and clinical response. In vitro pharmacology studies allow assessment of the potential contribution to the pharmacological profile by metabolites. Potency of dofetilide and metabolites has been compared for class III (K+ channel blockade) and class I (Na+ channel blockade) antidysrhythmic activities. Three of the metabolites of dofetilide displayed class III activity, but at concentrations at least 20-fold higher than dofetilide. Dofetilide N-oxide showed class I activity, but only at high concentration. Neither resting membrane potential or action potential amplitude were affected by any metabolite. This lack of biologically relevant activity is in accord with the close correlation between plasma concentrations of dofetilide and pharmacological response.

Action Potentials↗
PubMed1995

Excited state dynamics in photosystem I: effects of detergent and excitation wavelength.

Femtosecond transient absorption spectroscopy has been used to investigate the energy transfer and trapping processes in both intact membranes and purified detergent-isolated particles from a photosystem II deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803, which contains only the photosystem I reaction center. Processes with similar lifetimes and spectra are observed in both the membrane fragments and the detergent-isolated particles, suggesting little disruption of the core antenna resulting from the detergent treatment. For the detergent-isolated particles, three different excitation wavelengths were used to excite different distributions of pigments in the spectrally heterogeneous core antenna. Only two lifetimes of 2.7-4.3 ps and 24-28 ps, and a nondecaying component are required to describe all the data. The 24-28 ps component is associated with trapping. The trapping process gives rise to a nondecaying spectrum that is due to oxidation of the primary electron donor. The lifetimes and spectra associated with trapping and radical pair formation are independent of excitation wavelength, suggesting that trapping proceeds from an equilibrated excited state. The 2.7-4.3 ps component characterizes the evolution from the initially excited distribution of pigments to the equilibrated excited state distribution. The spectrum associated with the 2.7-4.3 ps component is therefore strongly excitation wavelength dependent. Comparison of the difference spectra associated with the spectrally equilibrated state and the radical pair state suggests that the pigments in the photosystem I core antenna display some degree of excitonic coupling.

Biophysical Phenomena↗
PubMed1994

Expression, purification, and characterization of the dihydrolipoamide dehydrogenase-binding protein of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitating its purification. The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence. The E3BP-E3 complex was highly purified and then separated into E3 and E3BP by chromatography on hydroxylapatite in the presence of 5 M urea. The E3BP-E3 complex combined rapidly with a pyruvate dehydrogenase (E1)-dihydrolipoamide acetyltransferase (E2) subcomplex (E1-E2 subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast. The stoichiometry of binding of E3BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E2 was determined with a truncated form of E2 (tE2, residues 206-454) lacking the lipoyl domain and the E1-binding domain, and with E1-E2 subcomplex, which contains intact E2. Mixtures containing tE2 or E1-E2 subcomplex and excess E3BP or E3BP-E3 complex were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or E3BP-E3, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that the E1-E2 subcomplex binds about 12 E3BP monomers attached to 12 E3 homodimers. Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C.

After many unsuccessful attempts to detect cDNA encoding the catalytic subunit of bovine pyruvate dehydrogenase phosphatase (PDPc) in bovine cDNA libraries, an approach based on the polymerase chain reaction (PCR) was undertaken. Overlapping DNA fragments were generated by PCR from bovine genomic DNA and from cDNA synthesized from total RNA with synthetic oligonucleotide primers on the basis of experimentally determined amino acid sequences. The DNA fragments were subcloned and sequenced. The complete cDNA is 1900 base pairs in length and contains an open reading frame of 1614 nucleotides encoding a putative presequence of 71 amino acid residues and a mature protein of 467 residues with a calculated M(r) of 52,625. Hybridization analysis showed a single mRNA transcript of about 2.0 kilobases. Comparison of the deduced amino acid sequences of the mitochondrial PDPc and the rat cytosolic protein phosphatase 2C indicates that these protein serine/threonine phosphatases evolved from a common ancestor. The mature form of PDPc was coexpressed in Escherichia coli with the chaperonin proteins groEL and groES. The recombinant protein (rPDPc) was purified to near homogeneity. Its activity toward the bovine 32P-labeled pyruvate dehydrogenase complex was Mg(2+)-dependent and Ca(2+)-stimulated and comparable to that of native bovine PDP. An active, truncated form of rPDPc, with M(r) approximately 45,000, was produced in variable amounts during growth of cells and/or during the purification procedure.

Amino Acid Sequence↗
PubMed1993

Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae.

The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1 beta exhibit 62% sequence identity. The size of the mRNA is approximately 1.5 kilobases. Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II. Disruption of PDH beta 1 is not lethal under vegetative growth conditions. The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Acetyltransferases↗
PubMed1992

Biochemical and molecular genetic aspects of eukaryotic pyruvate dehydrogenase multienzyme complexes.

The alpha-keto acid dehydrogenase multienzyme complexes play central roles in metabolism, are major sites of regulation, and are clinically important. Genes and cDNAs encoding the components of these complexes have been cloned and sequenced. Protein engineering and molecular cloning experiments are providing new insight into organization, structure-function relationships, and the molecular basis of genetic defects in these multienzyme complexes.

Acyltransferases↗
PubMed1991

Functional analysis of the domains of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

The LAT1 gene encoding the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae was disrupted, and the lat1 null mutant was used to analyze the structure and function of the domains of E2. Disruption of LAT1 did not affect the viability of the cells. Apparently, flux through the PDH complex is not required for growth of S. cerevisiae under the conditions tested. The wild-type and mutant PDH complexes were purified to near-homogeneity and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme assays. Mutant cells transformed with LAT1 on a unit-copy plasmid produced a PDH complex very similar to that of the wild-type PDH complex. Deletion of most of the putative lipoyl domain (residues 8-84) resulted in loss of about 85% of the overall activity, but did not affect the acetyltransferase activity of E2 or the binding of pyruvate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), and protein X to the truncated E2. Similar results were obtained by deleting the lipoyl domain plus the first hinge region (residues 8-145) and by replacing lysine-47, the putative site of covalent attachment of the lipoyl moiety, by arginine. Although the lipoyl domain of E2 and/or its covalently bound lipoyl moiety were removed, the mutant complexes retained 12-15% of the overall activity of the wild-type PDH complex. Replacement of both lysine-47 in E2 and the equivalent lysine-43 in protein X by arginine resulted in complete loss of overall activity of the mutant PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed1991

Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex.

Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Purification and properties of branched-chain alpha-keto acid dehydrogenase kinase from bovine kidney.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) kinase was purified 5000-fold to apparent homogeneity from extracts of bovine kidney mitochondria. The kinase co-purified with the BCKDH complex. About 70% of the kinase was released by treatment of the complex with 1.5 M NaCl and 0.1% 2-mercaptoethanol at pH 7.4, followed by chromatography on Sephacryl S-400. The uncomplexed kinase was purified further by chromatography on Q Sepharose and Superose 12. The purified kinase is a monomer of apparent Mr approximately 43,000. BCKDH kinase exhibited little activity, if any, toward pyruvate dehydrogenase.

Animals↗
PubMed1990

Autopsy-documented cure of multiple myeloma 14 years after M2 chemotherapy.

Multiple myeloma was diagnosed in a 65-year-old woman in 1974 who thereafter received five-drug M2 chemotherapy. All protein abnormalities subsequently returned to normal and serial bone marrow studies documented complete bone marrow remission. Destructive bone lesions persisted radiographically, but did not progress. In 1987, a localized sigmoid adenocarcinoma was resected. In 1988, the patient presented with multiple brain metastases associated with a primary pulmonary adenocarcinoma that proved rapidly fatal. At autopsy, no evidence of multiple myeloma was found. This report describes the first tissue-documented cure of multiple myeloma 14 years after diagnosis and initiation of M2 chemotherapy. The possible association of multiple myeloma with other malignancies is also discussed.

Aged↗
PubMed1990

Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

The inner core domain (residues approximately 221-454) of the dihydrolipoamide acetyltransferase component (E2P) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via the expression vector pKK233-2. The truncated E2p was purified to apparent homogeneity. It exhibited catalytic activity (acetyl transfer from [1-14C]acetyl-CoA to dihydrolipoamide) very similar to that of wild-type E2p. The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron. These findings demonstrate that the active site of E2p from S. cerevisiae resides in the inner core domain, i.e., catalytic domain, and that this domain alone can undergo self-assembly. The purified truncated E2p showed a tendency to aggregate. Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues approximately 181-220) to the catalytic domain. All dihydrolipoamide acyltransferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini. By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism [Guest, J. R. (1987) FEMS Microbiol. Lett. 44, 417-422]. Substitution of the sole His residue in the S. cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the kcat or Km values of the truncated E2p. However, the Asp-431----Asn, Ala, or Glu substitutions resulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kcat, with little change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed1990

Induction of ornithine decarboxylase in cerebral cortex by excitotoxin lesion of nucleus basalis: association with postsynaptic responsiveness and N-methyl-D-aspartate receptor activation.

The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal forebrain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injection results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the polyamines spermidine and spermine were unaffected by lesioning. Increases on ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK-801, a result indicating the involvement of N-methyl-D-aspartate (NMDA) receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase.

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E1 alpha; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a lambda gt11 cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E1 alpha. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E1 alpha. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E1 alpha and the 5' end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids (Mr = 42,703). Hybridization analysis showed that the size of the mRNA is about 1.4 kilobases.

Amino Acid Sequence↗