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Biomedical subjects

S M Boyle

Publications and source records attributed to S M Boyle.

At least 55 records · Page 3Linked to original sources
PubMed1990

Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

The DNA sequence of a 3.23-kilobase fragment of the Escherichia coli chromosome encoding biosynthetic arginine decarboxylase (ADC) was determined. This sequence contained the speA open reading frame (ORF) as well as partial speB and metK ORFs. The ADC ORF is 1,974 nucleotides long; the deduced polypeptide contains 658 amino acids with a molecular size of 73,980 daltons. The molecular weight and predicted ADC amino acid composition are nearly identical to the amino acid analysis of purified ADC performed by Wu and Morris (J. Biol. Chem. 248:1687-1695, 1973). A translational speA-lacZ fusion, pRM65, including 1,389 base pairs (463 amino acids) of the 5' end of speA was constructed. Western blots (immunoblots) with beta-galactosidase antisera revealed two ADC::beta-galactosidase fusion proteins in E. coli bearing pRM65: 160,000 and 156,000 daltons representing precursor and mature hybrid proteins, respectively. The predicted amino acid sequence of ADC contains a region of six amino acid residues found in two bacterial diaminopimelic acid decarboxylases and three eucaryotic ornithine decarboxylases. This conserved sequence is located approximately eight amino acids from the putative pyridoxal phosphate-binding site of ADC and is predicted to be involved in substrate binding.

Amino Acid Sequence↗
PubMed1988

Susceptibility of Microsporum and Trichophyton species to suicide inhibitors of polyamine biosynthesis.

DFMO (difluoromethylornithine) and DFMA (difluoromethylarginine), irreversible suicide inhibitors of ornithine and arginine decarboxylase activities (ODC and ADC) respectively, inhibit the growth of six species of Microsporum and six species of Trichophyton. Trichophyton species in general, are more sensitive than Microsporum species. DFMA inhibits growth as effectively as DFMO but at a 10-fold lower concentration. Inhibition is specific, as a number of substrates and end products of ODC and ADC activities antagonize DFMA and DFMO (i.e. ornithine greater than putrescine = spermidine greater than agmatine). The methylester analogue of ornithine, MFMOme (monofluormethyldehydroornithine methylester) is at least a 25-fold more effective inhibitor than DFMO; the inhibition is specific as it is reversed by ornithine.

Antifungal Agents↗
PubMed1987

Molecular mechanisms of the synergistic induction of ornithine decarboxylase by asparagine and glucagon in primary cultured hepatocytes.

In primary cultures of adult rat hepatocytes maintained in a salts/glucose medium, a more than 100-fold increase in ornithine decarboxylase (EC 4.1.1.17) activity was caused by asparagine and glucagon in a synergistic manner. The synthesis rate of ornithine decarboxylase was determined by [35S]methionine incorporation into the enzyme protein, and the amount of ornithine decarboxylase-mRNA was measured by hybridization with a cloned rat liver ornithine decarboxylase-cDNA. The synthesis rate of ornithine decarboxylase was stimulated more than 20-fold by asparagine and glucagon together, but the amount of ornithine decarboxylase-mRNA was increased only 3-4-fold, indicating that translational stimulation was involved in the induction process. Asparagine alone stimulated the synthesis of ornithine decarboxylase without substantial effect on the amount of ornithine decarboxylase-mRNA, whereas glucagon alone increased the amount of ornithine decarboxylase-mRNA about 3-fold without a detectable change in either enzyme activity or enzyme synthesis. Asparagine, at least in part, also suppressed degradation of ornithine decarboxylase.

Animals↗
PubMed1986

Transcription of the speC (ornithine decarboxylase) gene of Escherichia coli is repressed by cyclic AMP and its receptor protein.

The speC gene encoding ornithine decarboxylase (ODC) in Escherichia coli is negatively regulated by cAMP and the cAMP receptor protein (CRP). In minicells transformed with the plasmid pODC bearing speC, cAMP supplementation repressed ODC synthesis. In a cell-free protein synthesizing system directed by pODC, cAMP at 10(-5) M repressed ODC synthesis by 90%. This repression required a functional CRP as cAMP failed to repress ODC synthesis in vitro in an extract prepared from a crp- strain; the addition of purified CRP to the crp- extract restored the ability of cAMP to repress ODC synthesis. In a prototroph transformed with the plasmid pCOD bearing a speC::tet chimeric gene, cAMP supplementation decreased tetracycline (Tc) resistance. In contrast, in crp- strains transformed with pCOD or pTET (TcR), cAMP supplementation did not change their Tc resistance. When a cya- strain was supplemented with 2 mM cAMP, steady state levels of ODC mRNA were repressed by 80%. However, when a isogenic crp- strain was supplemented with 2 mM cAMP, no repression of ODC mRNA was observed. These results indicate that the cAMP-CRP complex exerts negative transcriptional control of ODC synthesis as a function of the speC promoter.

Cyclic AMP↗
PubMed1986

Purification and properties of agmatine ureohydrolyase, a putrescine biosynthetic enzyme in Escherichia coli.

The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (EC 3.5.3.11) catalyzes the conversion of agmatine to putrescine in Escherichia coli. AUH was purified approximately 1,600-fold from an E. coli strain transformed with the plasmid pKA5 bearing the speB gene encoding the enzyme. The purification procedure included ammonium sulfate precipitation, heat treatment, and DEAE-sephacel column chromatography. The molecular mass of nondenatured AUH is approximately 80,000 daltons as determined by gel-sieving column chromatography, while on denaturing polyacrylamide gels, the molecular mass is approximately 38,000 daltons; thus, native AUH is most likely a dimer. A radiolabeled protein extracted from minicells carrying the pKA5 plasmid comigrated with the purified AUH in both sodium dodecyl sulfate-polyacrylamide and native polyacrylamide gels. The pI of purified AUH is between 8.2 and 8.4, as determined by either chromatofocusing or isoelectric focusing. The Km of purified AUH for agmatine is 1.2 mM; the pH optimum is 7.3. Neither the numerous ions and nucleotides tested nor polyamines affected AUH activity in vitro. EDTA and EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] at 1 mM inactivated AUH activity by 53 and 74%, respectively; none of numerous divalent cations tested restored AUH activity. Ornithine inhibited AUH activity noncompetitively (Ki = 6 X 10(-3) M), while arginine inhibited AUH activity competitively (Ki = 9 X 10(-3) M).

Agmatine↗
PubMed1985

Biosynthetic arginine decarboxylase in Escherichia coli is synthesized as a precursor and located in the cell envelope.

The biosynthetic form of arginine decarboxylase (ADC) catalyzes the synthesis of agmatine, a precursor of putrescine, in Escherichia coli. Selective disruption of the cell envelope and an assessment of ADC activity or immunoprecipitable ADC in various fractions demonstrated its location between the cytoplasmic membrane and peptidoglycan layer. Expression in minicells of the speA gene encoding ADC resulted in the production of two immunoprecipitable species (74 and 70 kilodaltons). Studies in vivo with a pulse and chase of radiolabeled amino acid into the two species suggest a precursor-product relationship. This relationship was corroborated by demonstrating the accumulation of the 74-kilodalton species in a strain of E. coli unable to process signal sequences. Peptide mapping experiments with V8 protease, trypsin, and alpha-chymotrypsin demonstrated that the two species of ADC were very similar except for a minor difference. These data were used to substantiate the compartmentalization hypothesis as to how exogenous arginine can be channeled preferentially into putrescine.

Arginine↗
PubMed1984

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK).

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with Mrs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

Carboxy-Lyases↗
PubMed1984

Antagonistic transcriptional regulation of the putrescine biosynthetic enzyme agmatine ureohydrolase by cyclic AMP and agmatine in Escherichia coli.

The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (agmatinase; EC 3.5.3.11) catalyzes the conversion of agmatine to putrescine in Escherichia coli. The specific activity of AUH was determined in crude extracts prepared from wild-type strains and from strains with mutations in the adenylate cyclase gene (cya) or the cAMP receptor protein gene (crp) or both. In glucose minimal medium, a delta cya strain exhibited 70 to 90% higher AUH activity than a cya+ strain. Addition of 1 to 10 mM cAMP to cya+ and delta cya strains cultured in glucose repressed AUH activity in a dose-dependent manner. Addition of 1 to 10 mM cAMP to a delta crp strain failed to repress AUH activity. Addition of agmatine resulted in a three- to fourfold induction of AUH in delta cya and delta crp strains. This induction could be blocked by the addition of chloramphenicol. Simultaneous additions of various proportions of cAMP and agmatine resulted in reduced levels of induction and repression of AUH activity. This antagonistic regulation was shown to be exerted by independent mechanisms since AUH activity could be induced by agmatine in a delta crp strain supplemented with cAMP. These results suggest that both agmatine and cAMP antagonistically regulate AUH activity at the level of transcription. In minimal medium supplemented with 1 mM putrescine, the strains did not exhibit repression of AUH activity. In contrast, in minimal medium supplemented with 1 mM ornithine or arginine, cya+ or delta cya strains exhibited induced AUH activity as a result of conversion of these substrates to agmatine. Further experiments in vitro demonstrated that the effects observed with cAMP, agmatine, and arginine were not post-translationally mediated.

Agmatine↗
PubMed1984

Intergeneric homology of the speC gene encoding biosynthetic ornithine decarboxylase in Escherichia coli.

A 32P-labeled fragment of DNA containing the speC gene, which encodes the biosynthetic enzyme ornithine decarboxylase of Escherichia coli, was used as a hybridization probe for homologous sequences in the genomes of gram-negative and gram-positive bacteria. The speC probe detected homologous sequences in the DNA of only four members of the Enterobacteriaceae (Citrobacter freundii, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes); no homology was detected with the DNA of other representative members of the Enterobacteriaceae and gram-negative and gram-positive bacteria.

Enterobacteriaceae↗
PubMed1982

Negative control of ornithine decarboxylase and arginine decarboxylase by adenosine-3':5'-cyclic monophosphate in Escherichia coli.

The polyamine biosynthetic enzymes, ornithine decarboxylase (EC 4.1.1.17) (ODC) and arginine decarboxylase (EC 4.1.1.19) (ADC), are negatively controlled by cAMP in Escherichia coli. The specific activities of ODC and ADC were determined in crude extracts prepared from E. coli strains carrying a mutation in the adenylate cyclase (EC 4.6.1.1) structural gene (cya) and wildtype strains. These strains were cultured on various carbon sources in the presence and absence of cAMP. In wild-type strains, ODC and ADC activities were diminished in cells grown on glycerol compared to these strains cultured on glucose. When cya strains were grown on glucose or glycerol, ODC and ADC activities were the same. Addition of 1 mM cAMP to glucose-based medium repressed ODC and ADC activities in both the wild-type and cya strains. Furthermore, cAMP exerts its negative control through the cAMP receptor protein, since strains carrying a mutation in the crp structural gene fail to repress ODC and ADC activities in response to increased cAMP obtained by carbon source manipulation or cAMP supplementation of the growth medium. This evidence suggests that negative control of ODC and ADC by cAMP occurs at the level of transcription.

Acetates↗
PubMed1982

Biosynthetic ornithine and arginine decarboxylases: correlation of rates of synthesis with activities in Escherichia coli during exponential growth and following nutritional shift-up.

Whether guanosine tetraphosphate (ppGpp) has a role in the regulation of the putrescine biosynthetic enzyme, ornithine decarboxylase, in Escherichia coli is controversial. Different laboratories have reported either direct or indirect correlations between ppGpp levels and ornithine decarboxylase activity using different in vivo conditions. In this report, using conditions in vivo to modulate ppGpp levels, experiments are described which bear on the controversy. The rates of synthesis and biological activities of the biosynthetic ornithine and arginine decarboxylases (ODC and ADC) were measured in E. coli K-12 during experimental growth and during nutritional shift-up. There were good correlations between changes in their respective activities and the rates of synthesis of these enzymes during steady state or shift-up. ODC activity or rate of synthesis changed directly in concert with ppGpp levels, while ADC activity or rate of synthesis changed inversely with ppGpp levels. These observations support the contention that ppGpp does not inhibit ODC activity.

Carboxy-Lyases↗
PubMed1980

Synthesis of ribosomal protein S1 following nutritional shift-up in Escherichia coli K-12.

The synthesis rate of ribosomal protein S1 was measured in Escherichia coli K-12 during the transitional period following a nutritional shift-up from acetate minimal to glucose/amino acids/nucleosides medium. The synthesis rate of S1 increased without a lag suggesting that the S1 gene is under stringent control and located very close to its promoter. The rate of S1 synthesis slowed between 7 and 15 min, and then increased to the postshift-up steady state rate. The postshift-up steady state rate was half the initial rate obtained between 0 and 7 min. The slow down in the synthesis rate between 7 and 15 min indicates that an unknown factor(s), in addition to guanosine 5'-diphosphate, 3'-diphosphate, participates in the regulation of S1 gene expression.

Acetates↗
PubMed1980

Polyamines in encephalomyocarditis virus.

Encephalomyocarditis virus contains approximately 200 molecules of putrescine, 100 molecules of spermidine, and 40 molecules of spermine which could neutralize 11% of the viral genome. The same polyamines are present in different proportions in the Krebs ascites tumor cell in which the virus was grown.

Cadaverine↗
PubMed1979

Influence of magnesium and polyamines on the reactivity of individual ribosomal subunit proteins to lactoperoxidase-catalyzed iodination.

30S and 50S subunits, in the presence of either 20 mM Mg2+ or 6 mM Mg2+ and 5mM spermidine plus 25 mM putrescine, were observed to completely associate to form 70S monosomes as monitored by sucrose gradient sedimentation. Subunits maintained under the above ionic conditions were compared with 30S and 50S particles at low (6 mM) magnesium concentration with respect to the reactivity of individual ribosomal proteins to lactoperoxidase-catalyzed iodination. Altered reactivity to enzymatic iodination of ribosomal proteins S4, S9, S10, S14, S17, S19, and S20 in the small subunit of ribosomal proteins, L2, L9, L11, L27, and L30 in the large subunit following incubation with high magnesium or magnesium and polyamines suggests that a conformation change in both subunits accompanies the formation of 70S monosomes. The results further demonstrate that the effect of Mg2+ on subunit conformation is mimicked when polyamines are substituted for magnesium necessary for subunit association.

Iodoproteins↗
PubMed1978

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli.

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.

DNA-Directed RNA Polymerases↗
PubMed1977

Polyamine levels in Escherichia coli during nutritional shiftup and exponential growth.

At different exponential growth rates obtained either by varying the carbon source of the culture medium or limiting glucose uptake, intracellular levels of putrescine and spermidine were measured. Over a ten-fold increase in growth rate an approximately three-fold increase in putrescine level and a 3.5-fold increase in spermidine level per cell absorbance were observed. Conditions favoring an abrupt alteration in growth rate, such as occur following nutritional shiftup of Escherichia coli, resulted in a significant increase in the intracellular level of putrescine and virtually no change in the spermidine level. Because of the magnitude and the timing of the change in polyamine levels, the hypothesis that polyamines are (the components) responsible for inducing the rapid increase in the rate of RNA synthesis following nutritional shiftup is rejected.

Cell Division↗