Biomedical subjects

H Slegers

Publications and source records attributed to H Slegers.

At least 55 records · Page 3Linked to original sources

PubMed1986

Identification of the ribosomal proteins phosphorylated by the ribosome-associated casein kinase type II from cryptobiotic gastrulae of the brine shrimp Artemia sp.

Phosphorylation of the ribosomal proteins by the extra-ribosomal protein kinase was investigated "in situ" and with purified 40 S or 60 S ribosomal proteins from cryptobiotic embryos of Artemia sp. Ribosomal proteins that were most readily phosphorylated in 80 S ribosomes included S6 and S8 of the 40 S subunit and proteins L9, L13 and L18 of the 60 S subunit. Several additional polypeptides were phosphorylated when purified 40 S or 60 S ribosomal proteins were separately incubated in the reconstituted system. The possible functions of ribosomal phosphorylation in protein synthesis will be discussed.

Animals↗

PubMed1986

Identification of the substrates of the casein kinase II associated with non-polysomal messenger ribonucleoproteins of A. salina cryptobiotic embryos.

The association of a protein kinase with cytoplasmic non-polysomal messenger ribonucleoproteins is demonstrated by chromatography on oligo(dT)-cellulose and sucrose gradient centrifugation. The cAMP-independent enzyme is inhibited by caffeine and poly(L)-glutamic acid and is classified as a casein kinase II. Among the exogenous proteins initiation factor eIF2 is the best substrate and is 7.8 times more efficiently phosphorylated than casein. Endogenous mRNP protein substrates have a Mr of 125 000, 65 000, 38 000, 26 000 and 23 500. The main phosphate acceptor is the Mr 38 000 poly(A)-binding protein. Dephosphorylation of the poly(A)-binding protein by protein phosphatases decreases its RNA binding property. The effect of phosphorylation-dephosphorylation of mRNP proteins on the initiation of protein synthesis is discussed.

Animals↗

PubMed1985

Dephosphorylation of cytoplasmic non-polysomal messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina.

Cytoplasmic non-polysomal mRNP from cryptobiotic gastrulae of the brine shrimp Artemia salina do not contain endogeneous protein phosphatase activity. However, both non-polysomal mRNP and purified mRNP proteins, phosphorylated by mRNP associated protein kinase, can be dephosphorylated by protein phosphatases purified from A. salina cytosol and rabbit skeletal muscle. The 38 kDa and 23.5 kDa poly(A) binding proteins (P38 and P23.5) and a 65 kDa protein are the major substrates of each protein phosphatase used. The reversible phosphorylation-dephosphorylation of mRNP may be involved in the regulation of mRNP metabolism, by altering the poly(A) binding capacities of the mRNP proteins.

Animals↗

PubMed1985

Initiation factor eIF2 associated with non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina.

Non-polysomal poly(A)-containing mRNP of A. salina cryptobiotic embryos is separated in mRNP active in protein synthesis and in repressed mRNP by sucrose gradient centrifugation. In the translationally active fraction the presence of eukaryotic initiation factor 2 (eIF2) is demonstrated by electroblotting of sodium dodecylsulphate/polyacrylamide gels on nitrocellulose and anti-eIF2 antibody detection. mRNP proteins with Mr of 40 000 and 42 000 are identified as the alpha and beta subunits of eIF2. The repressed mRNP is devoid of eIF2 and is associated with an inhibitor ribonucleoprotein composed of a small 85 +/- 2-nucleotide-long RNA and a protein with Mr of 64 000. The latter ribonucleoprotein is a potent inhibitor of the translationally active mRNP.

Animals↗

PubMed1985

Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastrulae of the brine shrimp A. salina.

In the cytosol of A. salina cryptobiotic gastrulae at least five protein phosphatases active on phosphorylase a have been detected by ion exchange chromatography on DEAE-cellulose. Only two of these enzymes (PP-X and PP-Y) are active in mRNP dephosphorylation. Both enzymes are insensitive to inhibitor-1 and -2 and stimulation of enzymatic activity (2.5-fold with PP-X and 6.5-fold with PP-Y) can be accomplished by ethanol treatment of the native enzymes, or freeze-thawing in the presence of 1.7% (v/v) 2-mercaptoethanol. These properties allow PP-X and PP-Y to be classified as type-2A enzymes according to the nomenclature of Cohen. This paper is the first report of protein phosphatases capable of dephosphorylating mRNP proteins.

Animals↗

PubMed1985

Ribosome-associated cyclic nucleotide-independent protein kinase of Artemia salina cryptobiotic gastrulae.

An extra-ribosomal cAMP-independent protein kinase from cryptobiotic embryos of Artemia salina has been purified to near homogeneity by gel filtration on Bio-Gel A-0.5 m, ion-exchange chromatography on DEAE-cellulose and phosphocellulose P11 and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The enzymatic activity has a broad optimum at pH 7-8. Maximal activity is obtained in the presence of 5-6 mM MgCl2. The activity is inhibited by Mn2+, Ca2+ and K+. The enzyme has an Mr of 127 000, utilizes both ATP and GTP as phosphoryl donors and is completely inhibited by heparin and poly(L-glutamic acid). According to its properties, the enzyme can be classified as a casein kinase type II. Although the enzyme is associated with ribosomes, ribosomal proteins are not among the main substrates. The kinase is able to phosphorylate both the alpha and the beta subunits of initiation factor eIF2 using ATP or GTP as phosphoryl donors. The function of phosphorylation in the initiation of protein synthesis is discussed.

Animals↗

PubMed1985

Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina.

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina cryptobiotic gastrulae by ion-exchange chromatography on DEAE-cellulose, DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poly(A)-Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 mumol AMP . h-1 . (mg protein)-1. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94 000 and 70 000. The Mr-70 000 protein has been identified as poly(A) polymerase. The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH4+, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5'-triphosphates. AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration.

Animals↗

PubMed1984

Identification and properties of the 38 000-Mr poly(A)-binding protein of non-polysomal messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina.

The Mr-38 000 poly(A)-binding protein interacts with synthetic and natural RNA. A sequence-independent stoichiometry of one protein per 8 - 12 nucleotides is measured by filter binding and sucrose gradient centrifugation. Specificity for the poly(A) sequence is demonstrated from poly(A)/RNA mixing experiments. The poly(A)-binding protein has been identified as the helix-destabilizing protein HD40[Marvil, D. K., Nowak, L. and Szer, W. (1980) J. Biol. Chem. 255, 6466 - 6472] and is characterized by the existence of at least seven ionic species with a pI ranging from 9.2 to 6.6. Acidic ionic species are generated by phosphorylation with mRNP-associated protein kinase. Different ionic species are present on free mRNP and ribosomes-mRNP preinitiation complexes. The poly(A)-binding protein affects mRNA translation and (A)4 polyadenylation. The multifunctionality of the protein is discussed.

Animals↗

PubMed1983

Subcellular distribution in Xenopus laevis oocytes of a microinjected poly(A)-binding protein from Artemia salina gastrulae.

The poly(A)-binding protein P38 of non-polysomal mRNP from Artemia salina gastrulae was labelled by reductive methylation and microinjected into the cytoplasm of Xenopus laevis oocytes. The labelled protein has a half-life of approximately 20 h and accumulated in the nucleus of the oocyte. The kinetics of accumulation reached a plateau at about 15 h after microinjection. P38 accumulates in the nucleus to a final concentration 3.15 times higher than that reached by free diffusion. This fact suggests that P38, a cytoplasmic poly(A)-binding protein, might also play some role in the nucleus of the cell.

Animals↗

PubMed1982

The 38,000-Mr poly(A)-binding protein of non-polysomal messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina.

The 38,000-Mr poly(A)-binding protein has been purified to near homogeneity from non-polysomal messenger ribonucleoprotein of Artemia salina [Slegers, H., De Herdt, E., and Kondo, M. (1981) Eur. J. Biochem. 117, 111-120]. The protein consists of approximately 357 amino acids and is characterized by a high glycine content of 22.5% and the presence of dimethylarginine. From polynucleotide-protein binding experiments a stoichiometry of 9-11 adenylate and 10-12 uridylate residues per protein molecule is calculated. The polypeptide is devoid of poly(A) polymerase and RNase activities. The poly(A)-binding protein and the helix-destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472] have the same mobility in polyacrylamide/dodecylsulphate gel electrophoresis and exhibit a comparable amino acid composition and protein-polynucleotide stoichiometry. Based on the length of poly(A) sequences of mRNA and from protein-poly(A) binding experiments, a repetitive binding of the 38,000-Mr protein on the poly(A) sequence is demonstrated. The 38,000-Mr protein of cytoplasmic and membrane-bound non-polysomal messenger ribonucleoproteins is also compared.

Amino Acids↗

PubMed1981

A cryptobiosis-specific 19S protein complex of Artemia salina gastrulae.

The postribosomal supernatant of Artemia salina cryptobiotic embryos contains a large quantity of a 19S protein complex. An amount of 3.6 mg/g of cysts is measured by immunoprecipitation with anti-(19S protein complex) antibody. The quantity of this complex decreases during further development to nauplius larvae to only 15% of the quantity present in cryptobiotic embryos. The complex was no longer detectable after 7 days of growth. The 27000-Mr protein subunit of the 19S complex is not synthesized by mRNA isolated from cryptobiotic embryos. The cryptobiosis-specific complex has Mr 573000 and 610000 as calculated from light-scattering and sedimentation-diffusion measurements respectively. The 19S homocomplex contains 20-23 27000-Mr proteins and has no function in the translation of homologous mRNA. From hydrodynamic data a hydration of 1.25 g of water/g of protein is calculated. The abundant presence of the 19S protein complex in cryptobiotic embryos and the absence of synthesis during development to nauplius larvae indicate a functional role during the cryptobiotic process in early embryogenesis. A role in maintaining the water content of the cytoplasm above a critical threshold during desiccation is suggested.

Animals↗

PubMed1981

The use of Taka-diastase in a[3H]poly(A) hybridization assay of oligo(U) sequences in RNA.

A reliable assay of uridylate sequences longer than 10 is described. The procedure is based on the hybridization of [3H]poly(A) with poly(U) or oligo(U) sequences in high ionic conditions and a subsequent degradation of single stranded polynucleotides with purified Taka-diastase. A 1:2 complex between poly(A) and poly(U) is formed on which on poly(U) strand is digested by Taka-diastase. The procedure is especially suitable for the detection and quantitation of Un (n greater than 10) in RNA preparation.

Amylases↗

PubMed1981

Non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina.

Non-polysomal poly(A)-containing messenger ribonucleoprotein (mRNP) of Artemia salina has been isolated by thermal chromatography on oligo(dT)-cellulose in moderate (250 mM) and low (50 mM NaCl and 5 mM MgCl2) ionic strength. The purified particles sedimented between 5 S and 30 S and banded at a density of 1.38-1.40 g/cm3 and 1.26-1.27 g/cm3 in CsCl and sucrose isopycnic centrifugation, respectively. The translatability of the mRNP in a cell-free system depended on the conditions of isolation. The protein composition of the free mRNP is independent of the conditions used in oligo(dT)-cellulose chromatography. The proteins have Mr of 87,000, 76,000, 65,000, 50,000, 45,000, 38,000 and 23,500. A specific set of proteins is associated wtih different ribonucleoproteins, although some proteins are present on multiple particles. The main 17 +/- 2-S particle is composed of proteins with Mr of 87,000, 76,000, 45,000 and 38,000. Approximately the same proteins were present on free mRNP and mRNP isolated from non-polysomal mRNP-ribosome complexes. Poly(A)-binding proteins have Mr of 38,000 and 23,500. The 38,000-Mr protein comprised at least 60% of the total mRNP protein. Poly(A)-binding proteins with Mr of 38,000 and 76,000 are also present in a free state in the cytoplasm. A relation between the main poly(A)-binding mRNP protein and the helix-destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472] is discussed.

Animals↗

PubMed1980

Affinity chromatography of poly(adenylic acid)-containing ribonucleoproteins on oligothymidylic acid-cellulose.

Binding of poly(A)-containing RNP to oligo(dT)-cellulose has been investigated as a function of mono- and divalent ion concentration. 80-90% binding was obtained either in high (500 mM) or in moderate NaCl concentrations in the presence of 5 mM MgCl2. At 40 mM NaCl and 5 mM MgCl2 poly(A)+-RNP exhibit approximately the same stability as poly(A)+-RNA in binding to oligo(dT)-cellulose with a melting temperature of 41 and 45 degrees C, respectively, indicating that the protein moiety has no effect on the ribonucleoprotein binding in these conditions. Differences were observed in the elution of poly(A)+-RNA and poly(A)+-RNP from oligo(dT)-cellulose in buffer without salts. Poly(A)+-RNA was completely removed at 4 degrees C whereas the melting temperature of poly(A)+-RNP was only decreased to 34 degrees C. The isolation of poly(A)+-RNP by thermal elution from oligo(dT)-cellulose is described.

Animals↗

PubMed1979

Non-polyadenylated 22 s ribonucleoprotein particle is insensitive to translational inhibitor RNA of cryptobiotic gastrulae of Artemia salina.

A free cytoplasmic 22 S ribonucleoprotein particle exhibiting a major template activity in rabbit reticulocyte system has been identified in the cryptobiotic gastrulae of Artemia salina. This particle contains non-polyadenylated 9 S messenger RNA which codes primarily for a non-histone basic protein with an apparent molecular weight of 26 000 daltons. We have previously demonstrated the presence of a translational inhibitor RNA which is apparently responsible for transforming polyadenylated messenger (Slegers et al., FEBS Letters 80, 390-394, 1977). This inhibitor RNA was found to be completely ineffective on the template activity of non-polyadenylated 22 S messenger ribonucleoprotein, confirming the specificity of this regulatory RNA for polyadenylate sequences.

Animals↗

PubMed1979

Identification and characterization of a 19-S complex containing a 27 000-Mr protein in Artemia salina.

The cytoplasm of the cryptobiotic Artemia salina gastrula contains a large quantity of a unique 19-S complex. This particle is a specific aggregated form of a 27 000-Mr protein, having a molecular weight of 10(6) and an apparent buoyant density of 1.25 -- 1.26 g/cm3 in sucrose and 1.31 g/cm3 in CsCl. The relative quantity of this 19-S complex decreases significantly with respect to 80-S monoribosomes during the postgastrula development. Biochemical and immunological studies indicate that the 27 000-Mr protein is one of the RNA-binding proteins [Ovchinnikov et al., FEBS Lett. 88, 21 -- 26 (1978)] but is absent in the protein components associated with poly(A)-containing ribonucleoprotein complexes. The possibility is also suggested that the 27 000-Mr protein and Artemia elongation factor eEF-Ts might be related to each other on the basis of amino acid composition and immunological cross-reactivity.

Alkylation↗

PubMed1979

Characterization of protein components of poly(A)-containing messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina.

Conditions for the isolation of intact poly(A)+mRNP from cryptobiotic gastruale of A. salina are described. In the presence of Mg2+ ions nucleolytic cleavage occurs in vitro in the vicinity of the 3'-poly(A) segment of mRNP during the isolation procedure. The resulting two parts of poly(A)+mRNP complex are separated by thermal elution from oligo(dT)-cellulsoe affinity column. Analysis by SDS-gel electrophoresis of protein components associated with intact poly(A)+mRNP has revealed the existence of 20--30 S RNP complex containing five major proteins with Mr 68,000, 53,000, 50,000, 45,000 and 38,000, respectively, but completely lacking the poly(A)-specific Mr 76,000 protein.

Animals↗

PubMed1979

Regular, rodlike, macromolecular structures in Artemia salina.

Artemia salina of different origins and in different developmental stages contains regular, rodlike, macromolecular structures. The particles were visualized by electron microscopy after negative staining. They can be purified from the postmitochondrial supernatant by sucrose gradient centrifugation in the presence of EDTA. Their size distribution has been measured from electron micrographs. The particles are probably nucleoprotein complexes.

Animals↗