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C T Evans

Publications and source records attributed to C T Evans.

36 records · Page 2Linked to original sources
PubMed1988

Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase.

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Regulation of estrogen biosynthesis in human adipose stromal cells. Effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol esters on the synthesis of aromatase cytochrome P-450.

Using human adipose stromal cells in monolayer culture as a model system for study of the regulation of aromatase activity, as well as polyclonal antibodies raised in this laboratory against aromatase cytochrome P-450 (cytochrome P-450AROM), it was found that the rate of synthesis of cytochrome P-450AROM was stimulated by dibutyryl cyclic AMP. This stimulation was attenuated by epidermal growth factor and was potentiated by phorbol esters. These changes in cytochrome P-450AROM synthesis were associated with comparable changes in the levels of translatable cytochrome P-450AROM mRNA, as well as with changes in the activity of aromatase of these cells. By contrast, there was little change in the synthesis of the reductase component of the aromatase enzyme complex in response to these factors. The increase in mRNA was blocked by cycloheximide, indicative of a requirement for protein synthesis in mediating this inductive response. It is concluded that aromatase activity is regulated primarily by changes in the level of mRNA encoding cytochrome P-450AROM, and that such changes are likely a reflection of changes in the rate of transcription of the gene encoding this enzyme. Increases in the levels of cytochrome P-450AROM mRNA are apparently mediated by a regulatory protein(s), similar to that found for other steroidogenic forms of cytochrome P-450.

Adipose Tissue↗
PubMed1987

An improved method for protoplast formation and its application in the fusion of Rhodotorula rubra with Saccharomyces cerevisiae.

Protoplasts from various strains of red-pigmented yeasts were generated at high frequency using improved procedures. The use of sulphur-containing amino acids and 2-deoxyglucose in growth media led to impaired cell wall synthesis and rendered cells very susceptible to treatment with mercapto-ethanol and various lytic enzymes. Use of individual lytic enzymes separately resulted in relatively low frequencies of protoplasts from most of the red yeasts examined, whilst use of beta-glucuronidase, Novozyme and Zymolyase in series markedly increased stable protoplast formation. The latter effects were shown to be strain specific. The ability to generate large numbers of red yeast protoplasts prompted the attempt to examine intergeneric fusion between auxotrophs of a strain of Saccharomyces cerevisiae and Rhodotorula rubra. Putative hybrids were selected as variously-pigmented prototrophic colonies growing on minimal medium and stabilised by subculturing on the latter medium. Unusual cream, orange and yellow hybrid colonies were generated, composed of cells of varying morphologies (chains, multibudded). The majority of stable hybrids contained one nucleus, although several heterokaryons were also observed. Some hybrids possessed the phenotypes of both parents: fusant wcat41 grew as rapidly as the S. cerevisiae parent but also contained an inducible phenylalanine ammonia-lyase (PAL) which appeared to be more active than that of the Rhodotorula parent.

Culture Media↗
PubMed1987

Regulation of aromatase in estrogen-producing cells.

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EGF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a lambda gt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.

Adipose Tissue↗
PubMed1987

Sequencing of cDNA inserts encoding aromatase cytochrome P-450 (P-450AROM).

Two cDNA inserts complementary to mRNA encoding aromatase cytochrome P-450 (P-450AROM) have been isolated and characterized by restriction mapping and sequencing. The overlapping sequence encoded by these inserts is identical, and a putative heme-binding region has been identified. In addition, the open reading frame contains the sequences of all four cysteine-containing tryptic peptides isolated by Chen et al. (1986) from purified cytochrome P-450AROM. The inserts differ in the use of alternative poly A-addition signals, which is consistent with the presence of two major species of mRNA in human placenta, of 3.0 and 2.4 kb, which hybridize to these inserts. The identity of sequence between the two inserts and the likely presence of alternative poly A-addition signals, is suggestive that only one form of cytochrome P-450AROM is encoded by these mRNA species.

Amino Acid Sequence↗
PubMed1986

Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA.

A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene.

Adipose Tissue↗
PubMed1985

Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification.

Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.

Animals↗
PubMed1983

Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by adenine nucleotides.

The regulation of mitochondrial citrate metabolism has been investigated in oleaginous and non-oleaginous yeasts to ascertain its importance in controlling the rate of citrate efflux from mitochondria. The following observations were made: 1. Citrate efflux from mitochondria of the oleaginous yeast Candida curvata D, in the presence of L-malate and pyruvate, was stimulated by adding ATP and reduced by AMP. In the non-oleaginous yeast, Candida utilis 359, there was very little stimulation of citrate efflux by ATP but it was reduced by AMP. These effects appeared to be generalized as similar results were obtained in an examination of eight further yeasts (seven oleaginous and one non-oleaginous). 2. The effects of ATP and AMP were not observed in mitochondria whose metabolism had been inhibited by antimycin A and rotenone indicating the direct regulation of the citrate translocase was not involved. 3. In C. curvata D, ATP increased the total mitochondrial citrate content and reduced that of 2-oxoglutarate whereas AMP had the reverse effect. In C. utilis 359, AMP had a similar effect but that of ATP was much smaller. 4. To explain these observations the mitochondrial NAD+-dependent isocitrate dehydrogenase was studied in a number of yeasts. The enzyme from oleaginous yeasts had a requirement for AMP for activity and was inhibited by ATP. In non-oleaginous yeasts the enzyme was active in the absence of AMP and increased in activity as the isocitrate concentration increased. 5. The enzyme in C. curvata D was constantly more sensitive to increasing energy charge than that of the non-oleaginous yeast. These results indicate that the supply of citrate (and hence acetyl-CoA) to the cytosol is controlled by the activity of the intramitochondrial NAD+-dependent isocitrate dehydrogenase which in turn is regulated by adenine nucleotides. The sensitivity of this enzyme to the ATP/AMP ratio during lipogenesis is therefore an important control in the accumulation of lipid by yeasts.

Adenosine Monophosphate↗
PubMed1983

Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by long-chain fatty acyl-CoA esters.

1. Citrate efflux from a wide range of yeast mitochondria was inhibited by long-chain fatty-acyl-CoA esters. 2. Fatty-acyl-CoA esters with chain lengths of between 14 and 18 carbons were the most potent inhibitors of citrate efflux which was unaffected by the fatty acids themselves. 3. 50% inhibition of citrate transport was observed using palmitoyl-CoA and oleoyl-CoA at approx. 4-5 microM when the tricarboxylate carrier was saturated with L-malate as counter-anion. 4. The inhibition with palmitoyl-CoA and oleoyl-CoA was competitive with L-malate. 5. The possibility that the fatty-acyl-CoA esters were exerting their effect by acting as detergent was eliminated because of the low concentrations used and appropriate comparisons being made with non-specific detergents. Although detergents inhibited citrate efflux they also released citrate by causing membrane damage. 6. The effect of fatty-acyl-CoA esters on citrate efflux could be decreased by using higher mitochondrial protein levels and by adding bovine serum albumin. 7. The possibility is discussed that this inhibition represents a genuine feedback inhibition which could regulate the amount of lipid being synthesized by an oleaginous yeast.

Acyl Coenzyme A↗
PubMed1983

A comparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture.

The oleaginous yeast, Candida curvata D, was grown in both batch and continuous culture on 5 different carbon sources to compare the efficiency of fat production from the various substrates. Maximum lipid accumulation occurred in batch culture with xylose as the carbon source on nitrogen-limited medium reaching a level of 49% (w/w) of the biomass, but this was reduced to 37% at the optimum dilution rate (D = 0.05/hr) in a chemostat. Both the highest biomass and lipid yields were attained in continuous culture with lactose as the sole carbon source at a dilution rate of D = 0.04/hr, giving an efficiency of substrate conversion of 60 g of biomass and 18.6 g lipid per 100 g lactose utilized. The relative proportions of the major fatty acids (16:0, 18:0, 18:1, 18:2) in the lipid were found to vary considerably in batch culture and in continuous culture under carbon-limited conditions. However, on nitrogen-limited media in the chemostat, the fatty acid composition remained relatively constant over the whole range of dilution rates employed. Lipid from xylose-grown cells contained the greatest percentage of stearic acid (18:0) 15% and the lowest linoleic acid (18:2) 4%, whereas lipid from ethanol-grown cells contained elevated levels of oleic acid (18:1) 51% and decreased palmitic acid (16:0) 25%.

Candida↗
PubMed1983

Biochemical activities during lipid accumulation in Candida curvata.

Intracellular and extracellular concentrations of citrate and the specific activities of ten different enzymes in Candida curvata D were examined in relation to lipid biosynthesis in batch and continuous culture. Citrate was found to accumulate prior to lipid production and declined markedly as lipid accumulated in batch culture. The cells excreted citrate as the culture became nitrogen-limiting after 30 hr of growth, but little more was expelled after 40 hr when lipid accumulation was more marked. In continuous culture, only low levels of citrate were detected at the lower dilution rates and citrate was completely absent from both the cells and medium above a dilution rate of 0.1/hr. The activity of malic enzyme, malate dehydrogenase and ATP:citrate lyase increased in batch culture on lipid accumulated and, in continuous culture, both malic enzyme and ATP:citrate lyase varied in parallel with the specific rate of lipid synthesis which increased with increasing dilution rate. Activity of malate dehydrogenase, citrate synthase and glucose-6-phosphate dehydrogenase decreased with increasing dilution rate. The regulatory significance of these enzymes in lipid accumulation by C. curvata is discussed.

Ammonia↗
PubMed1980

Pyruvate phosphate dikinase: sequence of the histidyl peptide, the pyrophosphoryl and phosphoryl carrier.

Pyruvate phosphate dikinase contains a pivotal histidyl residue which functions to mediate the transfer of phosphoryl moieties during the reaction catalyzed by the enzyme. The tryptic peptide which contains this essential histidyl residue has been isolated by a two-step procedure originally developed by Wang and co-workers [Wang, T., Jurasek, L., & Bridger, W. A. (1972) Biochemistry 11, 2067]. This peptide has been sequenced by the manual dansyl-Edman procedure and is shown to be NH2-Gly-Gly-Met-Thr-Ser-His-Ala-Ala-Val-Val-Ala-Arg-CO2H. There is no readily interpretable homology between this peptide and other phosphorylated histidyl peptides previously isolated from other enzymes. By use of Chou & Fasman [Chou, P. Y., & Fasman, G. D. (1974) Biochemistry 13, 222], it is predicted that the sequence contains an alpha helix from the methionine residue through to the carboxyl terminal arginine residue.

Amino Acid Sequence↗
PubMedDate not supplied

Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types.

Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure. However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different. These divergences can be observed even between isoforms of the enzyme within the same species. Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur. Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2). A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells. Major differences were found between the hexameric citrate synthase originating from E. coli compared with those dimeric forms prepared from eukaryotic cells. Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases. Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2). These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast.

Animals↗
PubMedDate not supplied

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant.

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR. The oxidation of [1,2,3-13C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3-13C]propionate or [2-13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.

Allosteric Regulation↗