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Biosorption of cadmium and chromium (III) ions by means of selected yeast species has been estimated. Kinetics and equilibrium measurements have shown the reliable efficiency of both metals removal for Candida tropicalis. The influence of pH and ionic strength on biosorption process has been examined as well. For both metals the adsorption isotherms have been presented. The equilibrium of chromium (III) sorption has appeared compatible to Langmiur model and the maximum sorption capacity has been determined.

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The ribosomal stalk composed of acidic P1/P2 proteins and protein P0 is involved directly in the interaction of the elongation factors and mRNAs with the ribosome during protein synthesis. All P proteins are found to be phosphorylated in eucaryotic organisms. In Saccharomyces cerevisiae five different cAMP-independent protein kinases phosphorylating P proteins have been identified and characterized. In contrast to many other protein kinases, relatively little is known about inhibitors of these enzymes. A new protein inhibitor of protein kinases has been purified and characterized. It is a small (18.5 kDa) and acidic (pI = 4.2) protein with high inhibitory potency for PK60S and CK 2. The inhibitor is competitive with respect to protein substrates with Ki values in the range of approximately 6.5 microM for PK60S and approximately 22 microM for CK 2.

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A protein phosphatase dephosphorylating acidic ribosomal proteins was purified from Saccharomyces cerevisiae ribosome-free extract. It was shown that phosphoproteins from both P1 and P2 subfamilies as well as 60S "core" P0 protein were substrates for the enzyme. The phosphatase can dephosphorylate ribosomes as well as histones and casein but the two last substrates with significantly lower efficiency. It was found that the enzyme activity is Mn(2+)-dependent and inhibited by okadaic acid, tautomycin, cantharidin and nodularin at concentrations typical for protein phosphatase type 2A. The possible implications of those findings in the control of ribosome phosphorylation and therefore in the control of translation is discussed.

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Phosphorylation of ribosomal acidic proteins of Saccharomyces cerevisiae is an important mechanism regulating a number of active ribosomes. The key role in the regulatory mechanism is played by specific phosphoprotein kinases and phosphoprotein phosphatases. Three different cAMP-independent protein kinases phosphorylating acidic ribosomal proteins have been identified and characterized. The protein kinase 60S (PK60S), RAP kinase, and casein kinase type 2 (CK2). All three protein kinases phosphorylate serine residues which are localized in the C-terminal end of phosphoproteins. Synthetic peptides were used to determinate the amino acid sequence of phosphoacceptor site for PK60S. Peptide AAEESDDD derived from phosphoproteins YP1 beta/beta' and YP2 alpha turned out to be the best substrate for PK60S. A number of halogenated benzimidazoles and 2-azabenzimidazoles were tested as inhibitors of the three protein kinases. 4,5,6,7-Tetrabromo-2-azabenzimidazole inhibits phosphorylation only of these polypeptides phosphorylated by protein kinase 60S, namely YP1 beta/beta' and YP2 alpha, but not the other, YP1 alpha and YP2 beta phosphorylated by protein kinases RAP and CK2. RAP kinase has been found in an active form in the soluble fraction of S. cerevisiae. The enzyme uses ATP as a phosphate donor and is less sensitive to heparin than casein kinase 2. RAP kinase monophosphorylates the four acidic proteins. The ribosome-bound proteins are a better substrate for the enzyme. Multifunctional CK2 kinase phosphorylate all four acidic proteins. The kinase phosphorylates preferentially serine or threonine residues surrounded by cluster of acidic residues. The enzyme activity is stimulated in vitro by the presence of polylysine and inhibited by heparin.

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The phosphorylation sites of ribosomal acidic proteins (P proteins) from Saccharomyces cerevisiae were studied in vivo and in vitro by using CK-2, PK60S and RAP protein kinases. The three enzymes phosphorylate the last serine residues located in a highly conserved carboxyl end of the polypeptide chains. This was established by two-dimensional analysis of tryptic phosphopeptides from 32P-labelled proteins YP1 alpha, YP1 beta, YP2 alpha and YP2 beta, and by kinetic studies of the protein kinases with synthetic peptides corresponding to the fragments of endogenous ribosomal acidic polypeptides. In experiments with both endogenous P proteins and synthetic peptides as substrates protein kinase PK60S demonstrated unusual substrate specificity. In contrast to CK-2 and RAP protein kinases, PK60S phosphorylates predominantly two of the four P proteins, YP1 alpha and YP2 beta, with kinetic constants dependent on the primary structure of the N-terminal region of the polypeptide containing the target residue. The neutral amino acid, alanine, at position 3 in the peptide AAEESDDD (polypeptide fragments of YP1 beta and YP2 alpha) decreases the K(m) value more than 10-fold by comparison with the basic lysine residue at the same position in the peptide AKEESDDD (polypeptide fragments of YP1 alpha and YP2 beta).

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A new protein kinase, showing a high specificity for the ribosomal acidic P proteins (RAP kinase) has been purified and characterized from Saccharomyces cerevisiae extracts. Purification was carried out by four chromatographic steps, including DEAE-cellulose, phosphocellulose, heparin-Sepharose and P protein-Sepharose. The purified enzyme preparation contains only one polypeptide of around 55 kDa as determined by SDS gel electrophoresis and gradient centrifugation. RAP kinase is different from all previous well-characterized kinases and does not show cross-reaction with antibodies to the 71 kDa 60S ribosomal subunit-specific kinase PK60 previously reported. The enzyme uses ATP as a better phosphate donor and is less sensitive to heparin than casein kinase II but is moderately affected by salt. Among the different substrates tested, ribosomal acidic proteins are preferentially modified by RAP kinase, which phosphorylates only serine residues in the four P proteins as well as the related ribosomal protein P0. Casein is phosphorylated at a much lower level. All the data indicate that RAP kinase might be the enzyme responsible for the phosphorylation of the P proteins, and in this way may also participate in a possible translational regulatory mechanism.

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Several halogeno benzimidazoles and 2-azabenzimidazoles, previously shown to be relatively selective inhibitors of protein kinases CK-I and/or CK-II from various sources, including CK-II from yeast [Szyszka et al. (1995) Biochem. Biophys. Res. Commun. 208, 418-424] inhibit also the yeast ribosomal protein kinase PK60S. The most effective inhibitor of CK-II and PK60S was tetrabromo-2-azabenzimidazole ](TetraBr-2-azaBz), which was competitive with respect to ATP (and GTP in the case of CK-II) with Ki values of 0.7 microM for CK-II, and 0.1 microM for PK60S PK60S phosphorylates only three (YP1 beta', YP2 alpha) out of five polypeptides of pp13 kDa acidic proteins of 60S subunit phosphorylated by CK-II [Szyszka et al. (1995) Acta Biochim. Polon. 42, 357-362]. Accordingly, TetraBr-2-azaBz inhibits phosphorylation only of these polypeptides, catalysed by PK60S . Addition of TetraBr-2-azaBz to cultures of yeast cells, at concentrations which were without effect on cell growth, led to inhibition of intracellular phosphorylation of ribosomal acidic proteins, paralleling that observed in vitro. TetraBr-2-azaBz is shown to be a useful tool for studies on the intracellular regulation of phosphorylation of the ribosomal 60S acidic proteins, which are involved in formation of active ribosomes.

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Several halogeno benzimidazole riboside inhibitors of animal and plant protein kinases CK I and CK II (also known as casein kinases I and II), were found to be effective inhibitors of Saccharomyces cerevisiae CK II, but not of the 27-kDa CK.I or the 45-kDa CK I. The previously reported 5,6-dichloro-2-azabenzimidazole, which preferentially inhibits plant CK II relative to CK I, discriminates even more effectively between the yeast CK I and CK II enzymes. Two new analogues, tetrahalogeno-2-azabenzimidazoles, are even more potent inhibitors of CK II and much less so of CK I from yeast and animal sources. All inhibitors are competitive with respect to ATP (and GTP with CK II), the two latter with Ki values in the range 0.2-0.6 microM for CK II from yeast and mammalian sources.

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The native 80S ribosomes isolated from Saccharomyces cerevisiae (strain W303) cells was phosphorylated by two endogenous protein kinases: multifunctional casein kinase-2 (CK-2) and specific 60S kinase. Three acidic proteins within the 60S ribosomal subunit: YP1 beta, YP1 beta' and YP2 alpha are phosphorylated by both kinases. The other two proteins: YP1 alpha and YP2 beta are predominantly phosphorylated by CK-2 but not by 60S kinase. This was confirmed in the experiment with the recombinant protein, YP2 beta, as a substrate, which is practically not phosphorylated by specific 60S kinase. These results together with the previous data based on the target amino-acid sequences suggest that, in addition to the multifunctional casein kinase-2 and specific 60S kinase, there exist probably other protein kinase(s) which phosphorylate the ribosomal acidic proteins in the cell.

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Specific antibodies directed against the two yeast type-1 casein kinases (CK1) were used to study the localization of both 45 kDa and 27 kDa casein kinase species in yeast cells by immunofluorescence. Our results indicate that the larger and smaller CK1 species are localised in different compartments of the yeast cell. The 45 kDa enzyme is present in the cytoplasm of the cell both during the logarithmic and stationary growth phase. The 27 kDa CK1 was found in the nucleus in the cells in logarithmic growth phase while the enzyme from the stationary phase was present in the cytoplasm. Our results suggest that the 27 kDa casein kinase may play some role in yeast cell division control by displacement from the nucleus to the cytoplasm.

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Two yeast casein kinase type-1 species of 45 kDa and 27 kDa (CK1) were purified to apparent homogeneity and used for investigation of their immunological affinity. Antisera against the two kinases were isolated; the antibody against the 45 kDa kinase did not react with the 27 kDa enzyme. The 27 kDa casein kinase was recognized only by its own antibody. The obtained data strongly suggest that the low molecular mass CK-1 is not a proteolytic product of the 45 kDa kinase species.

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Elongation factor 2 (EF-2) of rabbit reticulocytes was phosphorylated in vitro by incubation with partially purified EF-2 kinase and [gamma-32P]ATP. After exhaustive tryptic hydrolysis 4 phosphopeptides were revealed by two-dimensional peptide mapping. The phosphopeptides were isolated by high performance liquid chromatography and sequenced. A comparison of the primary structure of the phosphopeptides with that of EF-2 showed that all 4 phosphopeptides originated from one region of EF-2 located near the N-terminus that contains 3 threonine residues: Thr-53, Thr-56, Thr-58. A direct estimation of localization of radioactive phosphate in the phosphopeptides demonstrated that all the enumerated threonine residues in EF-2 can be phosphorylated in vitro.

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A type 1 protein phosphatase from reticulocytes is shown to efficiently dephosphorylate the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase that phosphorylates the alpha subunit of eukaryotic peptide initiation factor 2, eIF-2. The kinase, activated in the presence of double-stranded RNA with concomitant phosphorylation of the Mr = 68,000 peptide, causes inhibition of peptide initiation and thereby effects translational control of protein synthesis. The Mn2+-dependent phosphatase is classified as a type 1 enzyme in that it is inhibited by inhibitor 2 in nanomolar concentrations and appears to have a Mr = 35,000 catalytic subunit. Dephosphorylation of the Mr = 68,000 peptide by the phosphatase is directly associated with a loss in kinase activity which can be restored by incubation with double-stranded RNA in the presence of ATP. The results demonstrate that the eIF-2 alpha kinase can undergo cyclic activation-inactivation that appears to be directly related to the phosphorylation state of the Mr = 68,000 peptide. They strongly support the previous conclusion that double-stranded RNA is required only for activation of the kinase and phosphorylation of the Mr = 68,000 peptide.

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The rabbit reticulocyte Mr 90,000 protein associated with the heme-sensitive eIF-2 alpha kinase has been identified previously as the mammalian heat shock protein of this size class (hsp 90). Purified reticulocyte hsp 90 when added exogenously to the kinase increases its activity. This stimulatory effect is abolished after incubation of hsp 90 with a highly purified type 1 phosphoprotein phosphatase isolated from reticulocytes. Phosphorylation of dephosphorylated hsp 90 by casein kinase II but not by cAMP-dependent protein kinase restores the biological activity of hsp 90 to stimulate eIF-2 alpha phosphorylation.

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Ser 51 in the NH2-terminal sequence of the alpha-subunit of eukaryotic peptide initiation factor 2 (eIF-2) has been identified as a second phosphorylation site for the heme-controlled eIF-2 alpha kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the alpha-subunit of spectrin. A synthetic peptide corresponding to eIF-2 alpha (41-54) is phosphorylated only in Ser 51 by the eIF-2 alpha kinase.

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Two protein kinases of Mr 43,000 and 23,000 from yeast, belonging to type-1 casein kinases, were purified to apparent homogeneity and used for investigation of their immunological affinity and for comparison of their peptide map patterns. The results obtained showed that antibodies against the 43 kDa kinase did not react with the 23 kDa enzyme. Moreover, the peptide maps of the radioiodinated kinases obtained either by chemical cleavage of peptide bonds in the presence of CNBr or by a limited digestion with V8 protease were completely different. All these observations point to the lack of relatedness between the two investigated enzymes.

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A type-2 casein kinase (YCK-2), lacking the 25-kDa autophosphorylatable beta subunit characteristic of animal casein kinases-2, has been obtained in a nearly pure form from Saccharomyces cerevisiae and was compared with liver casein kinase-2 (LCK-2). A 22-kDa phosphorylatable protein, copurifying with YCK-2, can be removed by ultracentrifugation at low ionic strength and is shown by several criteria to be unrelated to the beta subunit of LCK-2. The native Mr of YCK-2, deprived of the 22-kDa phosphoprotein, is about 150 000. Limited proteolysis experiments show that YCK-2 included 37-kDa catalytic subunits, which can be converted into still active 35-kDa proteolytic derivatives. These data are consistent with a homotetrameric quaternary structure as opposed to the heterotetrameric subunit composition alpha 2 beta 2 of LCK-2 and other animal casein kinases-2. Although many properties of YCK-2 and LCK-2, including substrate specificity, inhibition by heparin, polyglutamic acid and quercetin and stimulation by polyamines, are similar; their stability under denaturing and dissociating conditions and their response to polybasic peptides are quite different. In particular YCK-2 is more readily denatured than LCK-2 by heating and exposure to urea, sodium dodecylsulphate and deoxycholate while its activity is inhibited by 100-150 mM NaCl, which conversely stimulates LCK-2 activity 2-3-fold. The Km value of the synthetic peptide substrate Ser-(Glu)5 for YCK-2 is not significantly changed by the addition of polylysine. On the contrary the Km value of the same peptide substrate for LCK-2 decreases approximately tenfold upon addition of polylysine, which also prevents the fast autophosphorylation of the kinase at its beta subunit. These data suggest that the beta subunit of animal CK-2 may play a role in determining both the stability of the enzyme and its regulation and that, consequently, the different properties of YCK-2 may be at least in part accounted for by its lack of beta subunits.

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Casein kinase type II were isolated by the same procedure, from rat liver, human placenta, Querin carcinoma and yeast, and characterized. The mammalian enzymes were composed of three subunits alpha, alpha' and beta, whereas yeast kinase was composed of two subunits alpha and alpha'. It was shown that the catalytic activity, substrate and phosphate donor specificity, sensitivity to heparin and spermine were the same for all the kinases tested. The results give additional support to the suggestion [1] that the beta subunit is not required for optimal activity and specificity of yeast casein kinase II. The quaternary structure of the yeast enzyme of a molecular weight of approximately 150 000 is proposed as alpha2 alpha'2.

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