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Biomedical subjects

W F Anderson

Publications and source records attributed to W F Anderson.

At least 397 records · Page 22Linked to original sources
PubMed1971

Puromycin-peptide bond formation with reticulocyte initiation factors M1 and M2.

The ability to form a "peptide" bond between various forms of Met-tRNA or Phe-tRNA and puromycin has been studied in the reticulocyte cell-free system. When Met-tRNA(F), fMet-tRNA(F), or N-acetylPhe-tRNA are used as substrate at low Mg(++) concentration (3 mM), reticulocyte initiation factors M(1) and M(2) (M(2A) + M(2B)) are required for puromycin-peptide synthesis. In contrast to bacterial systems, this reaction is also stimulated by the elongation factor T(1). When Met-tRNA(M) or Phe-tRNA is used as substrate, there is no M-factor requirement for the puromycin reaction; T(1) is absolutely required, and the reaction is stimulated by T(2). These studies indicate that reticulocyte factors M(1) and M(2) may function in part by placing the initiator tRNA into the P site. The detailed mechanism for mammalian initiation, however, may be more complex than that for bacterial systems.

Cell-Free System↗
PubMed1971

Initial dipeptide formation in hemoglobin biosynthesis.

Initiation factors M(1) + M(2) from reticulocyte ribosomes bind Met-tRNA(F) to rabbit reticulocyte ribosomes containing endogenous hemoglobin mRNA. The initial binding of Met-tRNA(F) appears to be to the small ribosomal subunit. The Met-tRNA(F) is able to participate in what is presumed to be the first peptide bond in the formation of hemoglobin, namely the synthesis of a methionyl-valine dipeptide. The formation of this methionyl-valine dipeptide requires Met-tRNA(F), initiation factors M(1), M(2), and M(3), as well as Val-tRNA and T(1). No synthesis of methionyl-valine dipeptide takes place if Met-tRNA(F) is replaced by Met-tRNA(M), or if initiation factor M(3) is omitted. Thus, Met-tRNA(F) appears to be the initiator tRNA for hemoglobin biosynthesis and M(3), although required for the synthesis of the first peptide bond of hemoglobin, does not appear to be necessary, under the experimental conditions studied, for Met-tRNA(F) binding.

Animals↗
PubMed1971

Requirement for GTP in the initiation process on reticulocyte ribosomes and ribosomal subunits.

The requirement for GTP in the initiation process on reticulocyte ribosomes and ribosomal subunits has been examined by studying Met-tRNA(F) binding, ribosome-dependent [gamma-(32)P]GTP hydrolysis, and peptide-bond formation with puromycin. Met-tRNA(F) binding can be obtained with the methylene analogue, 5'-guanylylmethylene diphosphonate, as well as GTP, and it is not inhibited by fusidic acid or several other inhibitors of protein synthesis. This reaction can be performed with the 40S subunit and has the same requirements as the Met-tRNA(F)-binding reaction with washed ribosomes. Ribosome-dependent [gamma-(32)P]GTP hydrolysis can be obtained with the initiation factor M(2A) using either washed ribosomes or the 40S subunit. This reaction is also not significantly inhibited by fusidic acid. Peptide-bond formation between puromycin and Met-tRNA(F), however, is inhibited by fusidic acid, and does not occur if the methylene analogue of GTP is substituted for GTP. These data suggest that the binding of the initiator tRNA to the 40S subunit does not require the hydrolysis of GTP, but that at least one GTP hydrolysis event must occur after Met-tRNA(F) binding in order for the first peptide bond to be formed.

Anti-Bacterial Agents↗
PubMed1971

Isolation and translation of hemoglobin messenger RNA from thalassemia, sickle cell anemia, and normal human reticulocytes.

Human hemoglobin messenger RNA was isolated by sucrose gradient centrifugation from reticulocytes of patients having various hemolytic anemias. Using a messenger RNA-dependent cell-free system derived entirely from rabbit reticulocytes, the human hemoglobin messenger RNA has been translated and the products analyzed by carboxymethylcellulose column chromatography. Normal messenger RNA directs synthesis of normal human alpha- and beta-globin chains in nearly equal amounts. Sickle cell anemia messenger RNA directs the synthesis of normal alpha- and sickle beta-chains, beta-thalassemia messenger RNA directs the synthesis of normal alpha- and beta-chains, but the amount of beta-globin synthesized is markedly reduced. Thus the inability of the thalassemia reticulocyte to produce beta-globin is clearly attributable to the beta-globin messenger RNA.

Anemia, Sickle Cell↗
PubMed1970

Cell-free hemoglobin synthesis in beta-thalassemia.

Human ribosomes obtained from the reticulocytes of patients having either homozygous beta-thalassemia (thalassemia ribosomes) or a hematological disorder unrelated to thalassemia ("normal" ribosomes) have been utilized in a cell-free system highly active in the synthesis of intact human globin chains. This system is dependent on the addition of a ribosomal wash fraction from reticulocytes that contains factors necessary for chain initiation. In response to the ribosomal wash fraction, isolated from either thalassemia, normal human, or rabbit reticulocytes, normal human ribosomes synthesize equal amounts of alpha and beta chains. In contrast, in response to all three types of ribosomal wash fractions, thalassemia ribosomes synthesize 8-times more alpha than beta chains, a ratio similar to that produced in the intact cells of these patients. The molecular defect in beta-thalassemia, therefore, does not appear to be associated with initiation factors.

Anemia, Hemolytic, Autoimmune↗
PubMed1970

Research on ageing.

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Affective Symptoms↗
PubMed1969

The effect of tRNA concentration on the rate of protein synthesis.

Two in vitro protein-synthesizing systems derived from E. coli have been utilized to demonstrate that the concentration of a tRNA species can regulate the rate of translation of a messenger RNA. (a) The rate of poly-U-directed C(14)-phenylalanine incorporation into protein is stimulated by concentrations of tRNA(Phe) from 1.5 x 10(-8) M to 3.0 x 10(-6) M, the latter representing a tRNA(Phe)/70S ribosome ratio of 7. (b) The rate of translation of poly A,G in a S-30 protein-synthesizing system derived from E. coli is limited by the amount of tRNA(Arg) recognizing the codewords AGA and AGG present in the extract. Polypeptide synthesis can be stimulated in direct proportion to the amount of this tRNA(Arg) species added to the reaction mixture. A mechanism for regulating the rate of protein synthesis at the translational level may be the slowing of polypeptide chain propagation at certain codons due to the presence of rate-limiting tRNA species.

Arginine↗