PubMed Health⌕ Search

Biomedical subjects

B Safer

Publications and source records attributed to B Safer.

At least 91 records · Page 5Linked to original sources
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

Evidence for role of m7G5'-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M3.

7-methylguanosine 5'-monophosphate inhibits protein synthesis in a fractionated, messenger-dependent, reticulocyte cell-free system. This compound also inhibits binding of histone mRNA to reticulocyte ribosomes as well as interaction of VSV mRNA and histone mRNA but not EMC virus RNA with purified initiation factor IF-M3. These studies provide evidence that the role of 7-methylguanosine in the mechanism for initiation of eukaryotic mRNA translation may be related to specific recognition of mRNA by initiation factor IF-M3.

Animals↗
PubMed1976

Polypeptide chain initiation in eukaryotes: mechanism of formation of initiation complex.

Artemia salina ribosomal subunits and highly purified reticulocyte initiation factors (IF) are used to study the mechanism of formation of the puromycin-sensitive initiation complex Met-tRNAi-80S ribosome-AUG. A complex with equimolar amounts of 40S subunit, GTP, and Met-tRNAi is formed at low Mg2+ concentration with a requirement for IF-MP (homogeneous) but not AUG or other factors. An 80S complex is formed only upon the further addition of AUG, IF-M2A, and IF-M2B, but not of either factor alone. This complex contains no GTP or GDP. A 40S complex, which cannot be converted to an 80S one, is formed when the nonhydrolyzable analog GMPPCP is substituted for GTP. IF-M2A has no effect on the formation of this complex, but IF-M2B enhances its formation.

Animals↗
PubMed1976

Purification and characterization of two initiation factors required for maximal activity of a highly fractionated globin mRNA translation system.

Two additional initiation factors (IF-M4 and IF-M5) have been purified and characterized both physically and biologically. IF-M4 is active as a single polypeptide chain with a molecular weight of 48,000. In contrast, IF-M5 is active as a complex with a molecular weight of about 500,000 and consists of seven major and several minor polypeptide components. Analysis of IF-M5 in two polyacrylamide gel electrophoresis systems indicated that one of the major polypeptide chains of IF-M5 was the 35,000 dalton subunit of IF-MP. This analysis also revealed that IF-M2A, IF-M3, and elongation factor 2 were present as minor components. Both IF-M4 and IF-M5 are required to achieve maximal activity in an assay system dependent on exogenous globin mRNA, but neither factor has been observed to stimulate model reactions that utilize artificial templates [poly(U) or AUG].

Animals↗
PubMed1976

Competition between cellular and viral mRNAs in vitro is regulated by a messenger discriminatory initiation factor.

Encephalomyocarditis viral RNA has previously been shown to outcompete host cellular mRNA has for translation in vitro in crude and fractionated protein synthesizing systems. In the present communication it is shown that the competition is regulated by an initiation factor or complex of factors, and not the 40S initiation complex per se. The factor primarily involved is the murine equivalent of a component present in a partially purified preparation of rabbit initiation factor M3. Both the murine and rabbit factors are clearly messenger discriminatory.

Animals↗
PubMed1976

Coordination of citric acid cycle activity with electron transport flux.

Feedback control between flux through the phosphorylating electron transport chain and the coordination of flux through individual steps of the citric acid cycle have been investigated under a number of different conditions of substrate availability and workloads in the isolated perfused rat heart. The transition from substrate-free perfusion to perfusion with glucose and insulin with no change of workload was associated with increases in the pool sizes of citric acid cycle intermediates except for oxaloacetate, but with an initial imbalance of flux through individual steps in the cycle and transport of anions of the malate-aspartate cycle across the mitochondrial membrane. Flux through citrate synthase initially increased while that through alpha-ketoglutarate dehydrogenase decreased. Of the components of the malate-aspartate cycle, flux through the malate-alpha-ketoglutarate exchange was increased prior to that through the glutamate-aspartate exchange and intramitochondrial aspartate aminotransferase. These changes can be accounted for on the basis of known kinetic controls of the enzyme and transport steps in response to increased pyruvate, acetyl-CoA, and NADH delivery at an approximately constant rate of ATP turnover.

Adenine Nucleotides↗
PubMed1976

Contribution of tissue acidosis to ischemic injury in the perfused rat heart.

The isolated perfused working rat heart preparation has been used to study the effects of respiratory acidosis on myocardial metabolism and contractilly. Hearts were perfused with 5 mM glucose and 10(-2) U/ml of insulin in order to enhance metabolsim of glucose relative to that of fatty acids. After perfusion with Krebs bicarbonate medium at pH 6.6, hearts rapidly ceased performing external work and peak left ventricular pressure fell by 75% after 5 minutes. Oxygen consumption, rate of ATP generation and overall glycolytic flux also declined rapidly. After about 2 minutes of perfusion, the fall of glycolytic flux showed a partial reversal, which was largely accounted for by increased lactate production, so that glucose oxidation decreased further. The reversal of glycoltic flux could be accounted for by partial release of H+ inhibition of phospho-fructokinase by increased tissue levels of adenosine 5'-diphosphate (ADP), adenosine monophosphate (AMP) and P1 and decreased levels of adenosine triphosphate (ATP) and creatine phosphate. The increased proportion of glucose uptake converted to lactate together with an increase of the tissue lactate/pyruvate ratio could be accounted for by inhibition of the malate-aspartate cycle combined with tissue hypoxia. Lactate accumulated in the tissue as a result of a decreased permeability of the plasma membrane to lactate. Decreased oxygen delivery to the myocardium was caused by secondary constriction of the coronary vessels. In further experiments, the coronary flow was regulated by an external pump which delivered fluid at a controlled rate into the aortic cannula above the coronary arteries, and the degree of tissue hypoxia was monitored by measuring changes of pyridine nucleotide reduction state by surface fluorescence techniques. The effects of acidosis uncomplicated by possible hypoxia were compared directly with those produced by ischemic hypoxia. The effects of acidosis under these conditions were similar to those described above, and to those produced by ischemia. From these and other data it is concluded that the effects of ischemia are caused by a lowering of the intracellular pH, which decreases the rate of energy production relative to the rate of energy demand. However, it is suggested that the primary cause of the decreased peak systolic pressure with either acidosis or ischemia is not a result of a defect of energy metabolism, but is due to alteration of the calcium cycle of the heart. Possible causes of irreversible heart failure after prolonged ischemia are discussed.

Acidosis, Respiratory↗
PubMed1975

Binding of MET-TRNAf and GTP to homogeneous initiation factor MP.

Homogeneous initiation factor MP forms a stable complex with Met-tRNAf which binds to nitrocellulose filters in the absence of ribosomal subunits. Complex formation is rapid at 0 degrees and the rate of reaction is stimulated 20-fold by GTP when freshly prepared initiation factor MP is used. Under optimal assay conditions, a 1:1:1 stoichiometry for initiation factor MP, GTP, and Met-tRNAf is indicated, based on a molecular weight for initiation factor MP of 180,000. Kinetic analysis of ternary complex formation suggests an ordered reaction sequence with binding of GTP followed by binding of Met-tRNAf. However, binding of GTP appears to produce an unstable state which leads to rapid inactivation of initiation factor MP in the absence of Met-tRNAf. Formation of a stable binary complex of initiation factor MP and Met-tRNAf occurs in the absence of GTP. The binary complex cannot subsequently bind GTP. While storage of initiation factor MP at 0 degrees for several weeks has no effect on the rate or extent of Met-tRNAf binding in the presence of GTP, the rate of binary complex formation is increased 10-fold. The binary and ternary complexes appear to bind to 40 S ribosomal subunits with equal efficiency.

Animals↗
PubMed1975

Purification and physical properties of homogeneous initiation factor MP from rabbit reticulocytes.

Initiation factor MP was purified 1570-fold with 67% recovery of total activity present in 0.5 M KCl extracts of rabbit reticulocyte ribosomes. Initiation factor MP forms a ternary complex with Met-tRNAf and GTP or a binary complex with Met-tRNAf alone, the details of which are presented in the accompanying paper (Safer, B., Adams, S. L., Anderson. W. F., and Merrick, W. C. (1975) J. Biol. Chem. 250, 9076-9082). Initiation factor MP was homogeneous by the following criteria: (a) electrophoresis as a single band in gels of 5, 6, 7, 8, 9, and 10% acrylamide; (b) equilibration as a single band during isoelectric focusing; (c) sedimentation as a single symmetrical boundary during sedimentation velocity experiments; (d) linear plots of sedimentation equilibrium data; (e) symmetrical absorbance (at 280 nm) and activity profiles during DEAE-cellulose and Sephadex G-200 chromatography, and (f) symmetrical distribution of initiation factor MP during sucrose density gradient band sedimentation. The molecular weight of the initiation factor MP monomer (0.2 mg/ml) by low speed sedimentation equilibrium was 90,800. Calculations based on the Stokes radius and sedimentation velocity show the existence of relatively stable 90,000-dalton monomers or 180,000-dalton dimers at low (0.1 mg/ml) and high (9.75 mg/ml) concentrations of initiation factor MP, respectively. Electrophoresis in sodium dodecyl sulfate gels indicates that initiation factor MP monomer is composed of two noncovalently linked subunits with molecular weights of 52,000 and 34,000. Despite a relatively normal amino acid composition and an isoelectric point of 6.4, initiation factor MP behaves as a basic protein, eluting from phosphocellulose at 650 mM KCl (pH 7.9). Both ternary complex formation and methionyl-puromycin synthesis co-purify, indicating that a single protein is required for both activities.

Amino Acids↗
PubMed1975

Eukaryotic initiation complex formation. Evidence for two distinct pathways.

Two distinct pathways have been elucidated which lead to the formation of an AUG-dependent initiation complex. One pathway involves the use of initiation factor M1 (IF-M1) to promote AUG-dependent binding of the initiator tRNA to the 40 S subunit, followed by joining of the 60 S subunit in the presence of IF-M2A, IF-M2B, and GTP. The second pathway involves the IF-MP-directed binding of initiator tRNA to the 40 S subunit via a ternary complex of IF-MP-GTP-Met-tRNAf. This reaction does not require AUG codon. However, subsequent formation of an 80 S initiation complex (as determined by methionyl-puromycin synthesis) required AUG as well as IF-M2A, IF-M2B, and GTP. Since both pathways require the same complementary initiation factors (at the same level), it would appear that the only difference is the manner in which the initiator tRNA is bound to the 40 S subunit, either by IF-M1 or IF-MP. Examination of the requirements for endogenous mRNA-directed methionyl-puromycin synthesis indicates a greater difference between IF-MP and IF-M1 in that only IF-MP was capable of forming an 80 S initiation complex which was sensitive to puromycin.

Animals↗
PubMed1975

Comparison of fMet-tRNAf and Met-tRNAf from Escherichia coli and rabbit liver in initiation of hemoglobin synthesis.

A comparison has been made of the ability of the formylated and unformylated initiator tRNAs of Escherichia coli and rabbit liver to participate in a number of model reactions of protein synthesis. These reactions include: (a) formation of a ternary complex composed of the initiator tRNA, GTP, and initiation factor MP; (b) ApUpG-directed binding of the initiator tRNA to 40 S subunits with initiation factor Ml; (c) formation of the artificial dipeptide, methionylpuromycin; (d) formation of the natural initial globin dipeptide, methionylvaline; and (e) synthesis of sheep alpha and betaB-globin chains on reticulocyte polysomes from a type BB sheep. The results of these studies indicate that although the prokaryotic initiator tRNA species function efficiently in the partial reactions which involve only binding, the methionine donated by the prokaryotic tRNA is not incorporated efficiently into peptide linkage. This suggests that the initial high level of binding of the E. coli initiator tRNAs may be nonspecific, and that the structure of the tRNA itself is important for specific recognition by eukaryotic initiation factors. The effect of formylation on the effectiveness of the initiator tRNA is not clear; it reduces activity in ternary complex formation, does not affect ApUpG-directed binding to 40 S subunits, and increases the rate or extent of incorporation of methionine, or both, into methionylpuromycin and globin chains.

Animals↗
PubMed1975

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation.

In the presence of added double-stranded RNA or oxidized glutathione, protein synthesis in heminsupplemented reticulocyte lysates declines abruptly after 8-12 min of incubation at 30 degrees. The kinetics of amino-acid incorporation are very similar to those seen when lysates incorporation are very similar to those seen when lysates are incubated in the absence of added hemin. The inhibitory effects of double-stranded RNA (dsRNA) and oxidized glutathione (GSSG) are partially overcome by a homogeneous initiation factor, IF-MP, which also stimulates protein synthesis in hemin-deficient lysates. This factor is involved in the binding of Met-tRNAfmet to 40S ribosomal subunits during protein chain initiation. However, neither dsRNA alone nor GSSG alone significantly inhibits formation of [40S subunit-Met-tRNAf] complexes induced in reticulocyte lysates by dsRNA or GSSG involves one or more components present in the lysates but absent from the fractionated in vitro system. Such components may be related to the translational inhibitor that is active in hemin-deficient lysates.

Animals↗