Marta Gatica

Effect of metal ions on the activity of cascein kinase II from Xenopus laevis

CaSein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant α and β subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7–10 mM for Mg2+, 0.5–0.7 mM for Mn2+ and 1–2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence ofMg2+. The apparent K m values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 μM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and α subunit. Inhibition of the holoenzyme by 400 μM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the α subunit.

Isolation of threonyl adenylate-enzyme complex

Abstract The aminoacyl-RNA synthetases have been shown to carry out the following two-step reaction: 1. 1) amino acid + ATP + enzyme ⇌ aminoacyl-AMP-enzyme + PPi 2. 2) aminoacyl-AMP-enzyme + sRNA ⇌ aminoacyl-sRNA + AMP + enzyme Evidence for the formation of the intermediate aminoacyl-AMP-enzyme complex has been reported by several investigators ( Hoagland, 1955 , De Moss and Novelli, 1955 , Berg, 1956 ). Tryptophanyl adenylate (Karasek, et , al. , 1958) and seryl adenylate (Webster and Davie, 1961) were isolated in trichloroacetic acid supernatant fractions after incubation of amino acid and ATP with substrate amounts of the respective enzymes. The present communication describes the isolation of enzyme-bound threonyl adenylate and the capacity of this complex to transfer the threonine directly to sRNA.

Activity of the E75E76 mutant of the α subunit of casein kinase II from Xenopus laevis

The cDNA gene coding for the α subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys75 and Lys76, changing the charge distribution of a very basic sequence found in the α subunit. Expression of the mutated cDNA in a pT7‐7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. K m for casein or a model peptide substrate, nor in its interaction with the activating β subunit. Inhibition by quercetin and by 5,6‐dichloro‐1‐β‐d‐ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII αE75E76 mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.

Nucleic acids can regulate the activity of casein kinase II

Casein kinase II purified from nuclei of Xenopus laevis oocytes is inhibited by several specific nucleic acids. This kinase, the main phosphorylating activity of the oocyte nucleus, is markedly inhibited by poly U at 10 μg/ml, and this polymer is a competitive inhibitor of the phosporylation of the substrate casein (Kiapp 80 nM). M 13 phage ssDNA and unfractionated yeast tRNA also inhibit between 50 and 200 μg/ml. Poly C, poly A, poly AG, dsDNA and Escherichia coli rRNA do not alter activity significantly at similar concentrations. Inhibitions are reversed by RNase (poly U, tRNA) or S1 nuclease (ssDNA). Oocyte casein kinase I or rabbit cAMP‐dependent protein kinase are not inhibited by poly U at 200 μg/ml. The sensitivity of the casein kinase II to these inhibitors suggests a regulatory role for nucleic acids in nuclear phosphorylation reactions.

Copolymers of glutamic acid and tyrosine are potent inhibitors of oocyte casein kinase II

Polypeptides rich in glutamic acid are strong inhibitors purified from isolated nuclei of Xenopus laevis oocytes of casein kinase II. The presence of tyrosine in these peptides greatly enhances their inhibitory capacity. Using casein as a substrate, copolyglu:tyr (4:1) has an I 50 value of 20 nM, 250 fold lower than that of polyglutamic acid which is 5 μM. A similar large difference is observed when a synthetic peptide is used as substrate. The inhibition of copolyglu:tyr is competitive with casein and can be completely reversed by high ionic strength. The relative inhibitory capacity of the polypeptides tested, in descending order, is copolyglu:tyr (4:1) > copolyglu:tyr (1:1) > polyglu > copolyglu:phe (4:1) > copolyglu:ala ( > copolyglu:leu (4:1). The high affinity for tyrosine‐containing acid peptides is shared by rat liver and yeast casein kinase II so that it seems to be a general property of these enzymes.

Site‐directed mutants of the β subunit of protein kinase CK2 demonstrate the important role of Pro‐58

The following amino acids of the Xenopus laevis β subunit of protein kinase CK2 (casein kinase 2) were changed to alanine: Pro‐58 (β P→A); Asp‐59 and Glu‐60 and Glu‐61 (β DEE→AAA); His‐151–153 (β HHH→AAA). The last 37 amino acids of the carboxyl end were deleted (β Δ179–215). Stimulation of CK2α catalytic subunit activity was measured with casein as substrate and the following relative activities were observed: β P→A > β DEE→AAA ⪢ β WT > β HHH→AAA ⪢ β Δ179–215. The β DEE→AAA and β P→A were similar to β WT in reducing CK2α binding to DNA but β Δ179–215 was less active. The results indicate that both Pro‐58 and the surrounding acidic cluster play roles in dampening the activation of CK2α and that the carboxyl end of β is involved in the interaction with CK2α.

The presence of peptidyl transferase in wheat embryo ribosomes.

Abstract Extensively washed wheat embryo ribosomes require a supernatant factor and GTP in order to synthesize the model peptide N-acetylphenylalanylpuromycin from N-acetylphenylalanyl-tRNA and puromycin. The reaction is inhibited by fusidic acid, suggesting a requirement for translocation. The factor does not affect the binding of N-acetylphenylalanyl-tRNA to ribosomes. In the presence of 30 % ethanol, N-acetylphenylalanylpuromycin and N-formylmethionylpuromycin are synthesized by the wheat embryo ribosomes without requirements of added supernatant enzymes, GTP or poly U. This result indicates that the peptide transferase is present in the wheat embryo ribosomal particles.

Aminoacyl transfer from phenylalanyl-tRNA microinjected into xenopus laevis oocytes

Abstract Xenopus laevis oocytes were injected with [14C] phe-tRNA and the fate of the aminoacyl moiety was studied. The radioactive phenylalanine is gradually hydrolized off the tRNA once inside the cell. The rate of deacylation of the tRNA is not affected by inhibition of cellular protein synthesis by puromycin or cycloheximide. Part of the radioactive amino acid that leaves the tRNA (30 to 65%) is transferred directly into the oocyte nascent proteins as evidenced by the fact that its incorporation into proteins is not reduced by coinjection with a large excess of [12C] phenylalanine. Aminoacyl transfer from injected phe-tRNA into proteins is inhibited by puromycin and cycloheximide.

Evidence for in vivo compartmentation of phenylalanyl-tRNA ligase in amphibian oocytes☆

Abstract The in vivo activity of phenylalanyl-tRNA ligase of Xenopus laevis oocytes was assayed by measuring the esterification of microinjected yeast tRNA Phe with [ 14 C]phenylalanine added to the extracellular medium. The three enzyme substrates, ATP, phenylalanine, and tRNA Phe , are present in the in vivo assay at saturating concentrations as seen by the fact that microinjection into the cell of additional amounts of these compounds does not increase the quantity of [ 14 C]Phe-tRNA Phe formed. The in vivo activity of Phe-tRNA ligase in oocytes at several stages of development is less than 10% of the in vitro activity measured in homogenates of the same cells. The in vivo assay of Phe-tRNA ligase in oocytes that have been microinjected with this enzyme partially purified from X. laevis ovary shows that the enzyme is not inhibited by the cellular conditions. The conclusion drawn from these experiments is that a large fraction of the Phe-tRNA ligase present in oocytes is in a cellular compartment which is not available to the injected tRNA.

Studies on the Binding of Aminoacyl-tRNA to Wheat Ribosomes

The correct positioning of specific aminoacyl-tRNAs on the ribosome-mRNA complex is a key reaction in protein synthesis (translation). Although the intimate mechanism of the reaction is still mysterious, much information has been obtained about the components and products of aminoacyl-tRNA binding to ribosomes in bacterial systems (1). We know much less about how this process occurs in eukaryotic cells.