An inhibitory effect of cyclic nucleotide-independent protein kinases on protein synthesis in vitro.
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Cell-Free System↗
Biomedical subjects
W Kudlicki
Publications and source records attributed to W Kudlicki.
On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo.
Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.
Cyclic AMP↗
Evidence for a highly specific protein kinase phosphorylating two strongly acidic proteins of yeast 60 S ribosomal subunit.
Two distinct, cyclic AMP-independent protein kinase (ATP : protein photransferase, EC 2.7.1.37) from yeast have been isolated and highly purified. The first of the enzymes, protein kinase 1 A, phosphorylates casein and phosvitin, and its cellular protein substrate is unknown. The second enzyme, protein kinase 1 B, phosphorylates two strongly acidic proteins, L44 and L45, of the 60 S ribosomal subunit.
Adenosine Triphosphate↗
Isolation and properties of two protein kinases from yeast which phosphorylate casein and some ribosomal proteins.
Three fractions of protein kinase from postribosomal supernatant of Saccharomyces cerevisiae, active in phosphorylation of casein, were resolved on DEAE-cellulose. Two of these fractions: protein kinase 1 and protein kinase 3, were further purified about 1000 and 1800-fold respectively. The kinase 1 appeared to exist as a monomer with a molecular weight of 50 000 and utilized only ATP as phosphoryl donor. The protein kinase 3 was an aggregated form of enzyme with a molecular weight of above half a million and used both ATP and GTP for protein phosphorylation. Both isolated enzymes showed variations in respect to Michaelis constants, and inhibitory effects exerted by monovalent cations and nucleotide phosphates. The activity of the kinases was not affected by the presence of cAMP (adenosine 3':5'-monophosphate) or cGMP, however, only protein kinase 1 appeared to be a cAMP nucleotide-independent enzyme. Despite these differences both enzymes equally phosphorylated two strongly acidic proteins of the 60-S ribosome subunit, possibly related to L7, L12 of Escherichia coli.
Adenosine Triphosphate↗
Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast.
A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized. It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 X 10(-6)M and 5.5 X 10(-5)M, respectively. Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [gamma-32P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.
Guanosine Triphosphate↗
Proteins of yeast ribosomal subunits: number and general properties.
1. Saccharomyces cerevisiae at the early stationary phase of growth accumulate 80 S ribosomes, easily dissociating into subunits, which retain full activity in phenylalanine polymerization in vitro. A simplified and efficient technique for large-scale preparation of yeast ribosomal subunits is proposed. 2. Presence of 34 proteins in 40 S subunit and 42 proteins in 60 S subunit was demonstrated by two-dimensional acrylamide-gel electrophoresis. Both ribosomal subunits contain acidic proteins: three in 60 S and six or seven in 40 S subunit. It seems that two of them correspond to prokaryotic proteins, L7 and L12. The total number of yeast ribosomal proteins is similar to those obtained for other Eukaryota.
Centrifugation, Zonal↗
Ribosome-associated protein kinase from Saccharomyces cerevisiae.
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Arginine↗
Cotranslational folding of nascent proteins on Escherichia coli ribosomes.
Evidence is presented for cotranslational folding of rhodanese or ricin during its synthesis on Escherichia coli ribosomes. During transcription-translation, full-length but enzymatically inactive polypeptides accumulated as peptidyl-tRNA on the ribosomes. These polypeptides were activated and released by subsequent incubation with the bacterial chaperones and with release factor (RF-2). Coumarin was incorporated cotranslationally at the N-terminus of the nascent protein from fluorophore-S-Ac-Met-tRNAf. Changes in fluorescence indicated that DnaJ bound to the nascent proteins and to a fluorescently labeled synthetic peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese. This peptide also bound to 70S ribosomes or 50S subunits but not to 30S subunits. It inhibited activation and RF-2-dependent release of the full-length ribosome-bound rhodanese. A deletion mutant of rhodanese lacking the N-terminal 23 amino acids was not accumulated on the ribosome but was synthesized very efficiently. However, the protein that was formed was enzymatically inactive. DnaJ did not bind to this deletion mutant on ribosomes. We conclude that the chaperone-mediated reactions facilitate binding of the N-terminal sequence of nascent proteins to a specific site on 50S ribosomal subunits where it blocks release. The ribosome-bound protein undergoes chaperone-mediated reactions that are required for folding into an enzymatically active conformation.
Amino Acid Sequence↗