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W C Merrick

Publications and source records attributed to W C Merrick.

At least 109 records · Page 6Linked to original sources
PubMed1982

Polyamines inhibit the protein kinase 380--catalyzed phosphorylation of eukaryotic initiation factor 2 alpha.

Polyamines putrescine, spermidine, and spermine specifically inhibit the PK 380--catalyzed phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha). Since te PK 380--dependent phosphorylation of eIF-2 alpha inhibits the initiation or protein synthesis, the possibility exists that the polyamines enhance protein synthesis by inhibiting the phosphorylation of eIF-2 alpha by PK 380.

Adrenal Cortex↗
PubMed1982

Methylation of elongation factor 1 alpha from the fungus Mucor.

A basic protein from the dimorphic fungus Mucor racemosus, found to be highly methylated, is shown to be protein synthesis elongation factor 1 alpha. This protein is the most abundant protein in hyphal cells but is less abundant in yeast cells. It is post-translationally methylated with the formation of mono-, di-, and trimethyllysine at as many as 16 sites. Nearly 20% of the 44 lysine residues of elongation factor 1 alpha from mycelia are modified while those from sporangiospores are virtually unmethylated.

Amino Acids↗
PubMed1980

Site-specific phosphorylation if initiation factor 2 by three cyclic nucleotide-independent protein kinases.

The site-specific phosphorylation of initiation factor 2 (eIF-2) by three different cyclic nucleotide-independent protein kinases from rabbit reticulocytes was examined. The hemin-controlled repressor modified serine in the alpha subunit (Mr = 38,000), while casein kinase II and protease-activated kinase II phosphorylated serine residues of the beta subunit (Mr = 53,000). Under conditions of maximal phosphorylation, 1 mol of phosphate was incorporated into eIF-2 by each of the protein kinases. However, following treatment of eIF-2 with alkaline phosphatase, 2 mol of phosphate were added by casein kinase II, indicating residual phosphate was present on the beta subunit of purified eIF-2. The tryptic and chymotryptic peptides of the phosphorylated subunits were analyzed by two-dimensional peptide mapping involving thin layer electrophoresis followed by ascending chromatography. When eIF-2 was phosphorylated by the hemin-controlled repressor, three major chymotryptic and four tryptic phosphopeptides with molecular weights ranging from approximately 600 to 3000 were identified. When phosphorylation of eIF-2 beta was examined, two tryptic and two chymotryptic phosphopeptides were obtained after phosphorylation with casein kinase II and were different from the phosphopeptides observed following phosphorylation with protease-activated kinase II. This indicates that three distinct sites in the beta subunit were phosphorylated, two by casein kinase II and one by protease-activated kinase II.

Animals↗
PubMed1979

Role of eukaryotic initiation factor 5 in the formation of 80 S initiation complexes.

The function of eukaryotic initiation factor 5 (eIF-5) from rabbit reticulocyte lysate has been studied by sucrose gradient preparation of 40 S and 80 S initiation complexes. eIF-5 is required for transfer of initiator tRNA from 40 S preinitiation complexes to puromycin-reactive 80 S complexes. The transfer is dependent upon GTP hydrolysis and is associated with release of eIF-2 and eIF-3 from the 40 S subunit. The GTP-dependent loss of eIF-2 and eIF-3 is catalyzed by eIF-5 in the absence of 60 S subunits or when subunit joining is prevented by edeine, but not when GTP is replaced by GuoPP(NH)P. Unstable 40 S subunit . Met-tRNAf complexes generated by eIF-5 can form puromycin-reactive 80 S complexes when 60 S subunits are added in the absence of added GTP. In addition, kinetic evidence is presented that indicates GTP hydrolysis occurs prior to 80 S complex formation.

Animals↗
PubMed1979

Evidence that a single GTP is used in the formation of 80 S initiation complexes.

Evidence is presented that the GTP initially bound in ternary complex (Met-tRNAf.GTP.eukaryotic initiation factor 2 (eIF-2)) is the same GTP that is hydrolyzed to allow joining of a 40 S preinitiation complex with 60 S subunits. This evidence was obtained by two quite dissimilar techniques. The first was a kinetic analysis of AUG-directed methionyl-puromycin synthesis using either eIF-2 of eIF-2A to direct the binding of Met-tRNAf to 40 S subunits. The second technique was the isolation of 40 S preinitiation complexes by Sepharose 6B chromatography and subsequent quantitation of GTP hydrolysis and methionyl-puromycin synthesis under conditions where 80 S complex formation is permitted.

Animals↗
PubMed1979

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.

Animals↗
PubMed1979

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.

Animals↗
PubMed1978

A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA.

Protein synthesis initiation factors prepared from rabbit reticulocyte and mouse ascites ribosomes were tested for the ability to crosslink to the 5' cap of mRNA. Crosslinking of one polypeptide of apparent molecular weight 24,000 was inhibited by the cap analogs, m7GMP and m7GDP, indicating a specific interaction with the cap. Although specific crosslinking of the 24,000 molecular weight polypeptide was found with eukaryotic initiation factor 3 and to a lesser extent with initiation factor 4B, both of these factors contained less than stoichiometric amounts of this polypeptide. The crosslinking method provides a highly sensitive and specific assay for cap-binding proteins and should facilitate their purification for functional studies.

Chromatography, DEAE-Cellulose↗
PubMed1978

Effect of hemin on site-specific phosphorylation of eukaryotic initiation factor 2.

Initiation factor 2 (eIF-2) is phosphorylated in vitro by two different cyclic nucleotide-independent protein kinases. As previously shown, a protein kinase activity that comigrates with the major casein kinase activity from rabbit reticulocytes phosphorylates eIF-2beta. In addition, a second protein kinase that specifically phosphorylates eIF-2alpha has been identified. Both protein kinase activities demonstrate cyclic nucleotide-independent activity and are not inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase activities. Phosphorylation of eIF-2alpha is almost completely inhibited by 20--35 muM hemin, whereas phosphorylation of eIF-2beta is only partially inhibited. Hemin acts by decreasing the rate of incorporation of phosphate into eIF-2alpha. The protein kinase activity that modifies eIF-2alpha has been shown to have inhibitory activity in the cell-free protein-synthesizing system, whereas the protein kinase for eIF-2beta has no effect. The identity of the former enzyme with the hemin-controlled repressor and role of hemin in the control of initiation are discussed.

Adenosine Triphosphate↗
PubMed1976

Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta.

The active protein components of initiation factor M2B (IF-M2B) have been resolved into two homogeneous factors. These proteins, IF-M2Balpha and IF-M2Bbeta, were purified 300- and 500-fold, respectively, with a yield of about 15% of the original starting activity. The low molecular weight (approximately 17,000) of these two proteins is in contrast with the much greater molecular weights that have been found for other initiation factors. IF-M2Balpha is also unique among the initiation factors in that it contains no tryptophan and is capable of self-association. Both proteins are required for model assays which utilize 40 S and 60 S subunits (poly(U)-directed polyphenylalanine synthesis or AUG-directed methionyl-puromycin synthesis). IF-M2Bbeta has been shown to be required for hemoglobin synthesis, however, the presence of high concentrations of IF-M2Balpha in the 100,000 X g lysate supernatant has precluded the demonstration of an IF-M2Balpha requirement in hemoglobin synthesis.

Amino Acids↗