Effects of erythropoietin on uridine metabolism in cell cultures of fetal calf liver

1984 ◽  
Vol 62 (2-3) ◽  
pp. 143-149 ◽  
Author(s):  
L. F. Congote
Keyword(s):  
Cell Cultures ◽  
High Performance ◽  
Specific Activity ◽  
Control Cell ◽  
Fold Increase ◽  
Reversed Phase ◽  
Uridine Incorporation ◽  
Soluble Cell ◽  
Cell Extracts ◽  
Chain Synthesis

The effect of sheep plasma erythropoietin preparations on the incorporation of [5-3H]uridine into erythroid cells has been studied using cells of fetal calf liver cultured in serum-free medium. The cells were incubated for 20 h with the hormone, followed by a 1-h incubation with [3H]uridine. Erythropoietin caused a 2.5-fold increase in the incorporation of uridine into cold trichloroacetic-acid-insoluble cell extracts and a 70% increase in the incorporation of uridine into the cold acid-soluble cell extracts. The phosphorylated metabolites of labeled uridine present in the cold acid-soluble fraction were analyzed by anion-exchange high performance liquid chromatography (HPLC). Erythropoietin increased the amounts of labeled UDP and UTP per cell. However, the specific activity of UTP and the labeled amounts of UDP-glucose in erythropoietin-treated cells were not significantly different from those in control cell cultures. After chromatography of the crude erythropoietin preparations on reversed-phase and gel-permeation HPLC, there was a perfect coincidence of the fractions stimulating uridine incorporation into acid-soluble and acid-insoluble cell extracts. The protein fractions from crude erythropoetin which stimulated uridine incorporation after purification by reversed-phased HPLC were also able to stimulate globin chain synthesis in fetal calf liver cells. These experiments suggest that the multiple effects on uridine metabolism described above are due to erythropoietin, rather than other proteins contaminating the crude hormone preparations.

Two-Component Anti-Staphylococcus aureusLantibiotic Activity Produced by Staphylococcus aureusC55

1998 ◽  
Vol 64 (12) ◽  
pp. 4803-4808 ◽  
Author(s):  
Maduwe A. D. B. Navaratna ◽  
Hans-Georg Sahl ◽  
John R. Tagg
Keyword(s):  
Specific Activity ◽  
Micrococcus Luteus ◽  
Fold Increase ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Bacteriocin Activity ◽  
Terminal Amino Acid ◽  
Molecular Weights ◽  
Terminal Amino ◽  
Distinct Peptide

ABSTRACT Staphylococcus aureus C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C8 and C18) chromatography. The yield following C8 chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill S. aureus or Micrococcus luteus but not S. epidermidis strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.

Aspartic Proteinase in Dugesia tigrina (Girard) Planaria

2002 ◽  
Vol 57 (5-6) ◽  
pp. 541-547 ◽  
Author(s):  
Fanny B. Zamora-Veyl ◽  
Herbert L. M. Guedes ◽  
Salvatore Giovanni-De-Simone
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
Ph Value ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Fold Increase ◽  
Maximal Activity ◽  
Single Step ◽  
Dugesia Tigrina ◽  
Final Yield

A proteolytic activity was identified in Dugesia tigrina planaria using the chromogenic substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP. The activity of the enzyme increased four times during the regeneration and presented a maximum at 120 hr being higher in tail than head regenerating segments. The protease that displays this activity was purified from worms by a single step on pepstatin-agarose followed by gel-filtration high performance liquid chromatography. The purification resulted in a 34-fold increase in specific activity and the final yield was 10%. The active D. tigrina hydrolase appears to be a dimeric protein composed of identical subunits with 34 kDa associated by disulphide bridges similar to vertebrate cathepsin D. By SDS-PAGE several bands were detected but upon gel filtration HPLC one proteolytically active component, termed Asp-68, was detected and isolated. The maximal activity was observed in a range between pH 3.5-5.0 and the enzyme became inactivated at a pH value above 7.2. The purified enzyme was not inhibited by inhibitors from serine (aprotinin, TPCK, PMSF and TLCK), metallo (EDTA) and cysteine proteinase (E-64) classes. In contrast, inhibitors such as pepstatin, EPNP, and 4-β-PMA efficiently inhibited the activity of the 68-kDa protease.

scholarly journals Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

1993 ◽  
Vol 4 (1) ◽  
pp. 79-92 ◽  
Author(s):  
L Connell-Crowley ◽  
M J Solomon ◽  
N Wei ◽  
J W Harper
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Fold Increase ◽  
Cyclin A ◽  
Cyclin B ◽  
Activation Process ◽  
Cyclin Dependent Kinase ◽  
Cell Extracts

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

Heterosis and the metabolism of [3H]gibberellin A1 in maize

1986 ◽  
Vol 64 (9) ◽  
pp. 2160-2164 ◽  
Author(s):  
S. B. Rood
Keyword(s):  
Retention Time ◽  
High Performance ◽  
Zea Mays L ◽  
Specific Activity ◽  
High Specific Activity ◽  
Reversed Phase ◽  
Single Cross ◽  
Parental Inbred ◽  
Labelled Compound ◽  
Gibberellin A1

Seedling growth and the metabolism of high specific activity (1.1 TBq mmol−1) [3H]gibberellin A1 were studied in two Canadian maize (Zea mays L.) inbreds, CM7 and CM49, and in their single cross F1 hybrid. As previously reported, the hybrid grew more rapidly than either parental inbred. Metabolism of the [3H]GA1 was qualitatively similar in all three genotypes and [3H]-labelled metabolites were tentatively identified through sequential, step-elution silicic acid partition chromatography followed by reversed-phase C18 high-performance liquid chromatography, relative to the retention times of authentic GAs. Although the expected 2β-hydroxylation product, [3H]GA8 was not detected, a metabolite was observed at the retention time of GA8-O(2)-glucoside. Additionally, another metabolite(s) eluted at the retention time of glucosyl conjugates of GA1, and enzymic cleavage with cellulase yielded a [3H]-labelled compound which subsequently eluted at the retention time of free GA1. While the ratio of the precursor [3H]GA1 to total [3H]GA conjugate-like metabolites was similar in the three genotypes, the hybrid contained higher levels of the [3H]GA8-glucosidelike metabolite, whereas the inbreds contained higher levels of the [3H]GA1 conjugatelike metabolite(s). Thus, there are differential rates of GA1 metabolism in a maize hybrid relative to its slower-growing inbred parents.

scholarly journals Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies

2002 ◽  
Vol 184 (7) ◽  
pp. 2019-2029 ◽  
Author(s):  
F. G. Healy ◽  
S. B. Krasnoff ◽  
M. Wach ◽  
D. M. Gibson ◽  
R. Loria
Keyword(s):  
Cytochrome P450 ◽  
Culture Filtrate ◽  
High Performance ◽  
Reversed Phase ◽  
Nucleotide Sequence Analysis ◽  
Cyclic Dipeptide ◽  
P450 Monooxygenase ◽  
Thaxtomin A ◽  
Cell Extracts ◽  
Streptomyces Acidiscabies

ABSTRACT The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3′ of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as 1H- and 13C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(l-4-nitrotryptophyl-l-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.

Adenine salvage enzymes and intracellular nucleotide triphosphate content in Physarum flavicomum amoebae during growth and development

1989 ◽  
Vol 35 (5) ◽  
pp. 554-558 ◽  
Author(s):  
Arunthathi Cumaraswamy ◽  
Henry R. Henney Jr.
Keyword(s):  
Formation Control ◽  
Hplc Analysis ◽  
Specific Activity ◽  
Fold Increase ◽  
Direct Conversion ◽  
Atp Content ◽  
Adenine Phosphoribosyltransferase ◽  
Predominant Role ◽  
Cell Extracts ◽  
Major Route

The specific activity of adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) and adenosine phosphorylase (EC 2.4.2.-), two enzymes involved in the utilization of exogenous adenine, was measured in extracts of myxamoebae-swarm cells of Physarum flavicomum undergoing growth, microcyst formation (control), and during adenine inhibition of encystment. Both enzymes showed a higher specific activity in adenine-inhibited cells (AIC) compared to normal control (NC) or growing cells (GC). These experiments revealed that the specific activity of APRT was 7.1-, 5.3-, and 1.7-fold higher than that of adenosine phosphorylase in AIC, GC, and NC, respectively. This suggests a predominant role for the enzyme APRT in the salvage of adenine in this organism. The major route for the utilization of adenine thus seems to be by its direct conversion to AMP rather than via its riboside adenosine. HPLC analysis of the ribonucleotide triphosphates in cell extracts of GC, NC, and AIC revealed a 2.6- and a 3.3-fold increase in the ATP and GTP content, respectively, in the AIC compared with the NC cells. The ATP content in the GC was higher by a factor of 2.2 compared with the NC cells, while the GTP content in the GC was only 0.6 times that in the NC cells. UTP levels in AIC and GC were 1.3- and 1.4-fold higher than in the NC cells. In contrast, the CTP level in AIC was lower than in NC cells and was not detectable in the growing cells.Key words: adenine phosphoribosyltransferase, ATP, GTP, amoebae, Physarum.

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