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Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes.

ABSTRACT: The effects of an affinity-purified polyclonal antibody to Artemia salina ribosomal protein L5 on protein synthesis in vitro were examined. The antibody interacted with 60 S subunits more strongly than with 80 S ribosomes, and inhibited reassociation of ribosomal subunits to some extent at 5 mM-Mg2+ but not at 10 mM. Polyphenylalanine synthesis in vitro at 10 mM-Mg2+ was significantly inhibited, especially when the antibody was first preincubated with 60 S subunits prior to the assay. The incorporation of amino acid directed by globin mRNA was inhibited only when the preincubation with 60 S subunits was carried out. On the other hand, no effect was detected on elongation factor 2- and 60 S subunit-dependent uncoupled GTPase activity. These results suggest that L5 is probably located at or near the subunit interface and may play an important role in protein synthesis.

SUBMITTER: Kenmochi N

PROVIDER: S-EPMC1138501 | biostudies-other | 1989 Apr

REPOSITORIES: biostudies-other

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Project description:The oxygen-binding characteristics of the three extracellular haemoglobins of brine shrimp (Artemia salina) were studied in vitro by using highly purified preparations. Haemoglobin I is induced last in the development of brine shrimps when functional gills are formed. It has the lowest oxygen affinity (p(50) 5.34mmHg), an intermediate Bohr effect (ø -0.09 at 20 degrees C) above pH8 and a temperature-sensitivity (DeltaH -44.8 to -45.6kJ/mol at pH8-9) comparable with those observed with other invertebrate haemoglobins [Weber & Heidemann (1977) Comp. Biochem. Physiol. A57, 151-155]. Haemoglobin II, which is the first to be induced, soon after hatching of nauplius larvae, persists generally throughout the whole adult life. It has an intermediate oxygen affinity (p(50) 3.7mmHg), the highest Bohr effect (ø -0.21 at 20 degrees C) above pH8 and a similar temperature-sensitivity (DeltaH -46.0 to -54.8kJ/mol at pH8-9) as haemoglobin I. However, haemoglobin III, which is induced second several hours after the induction of haemoglobin II but disappearing from the haemolymph in the middle of adult life, has the highest oxygen affinity (p(50) 1.8mmHg), the lowest Bohr effect (ø -0.03 at 20 degrees C) above pH8.5 and a high resistance against temperature variation between 10 and 25 degrees C at pH8.5-9 (DeltaH -22.6 to -23.0kJ/mol). At pH7.5-8, haemoglobin III exhibits a similar temperature-sensitivity under 30 degrees C as do other haemoglobins. All three haemoglobins have a rather low co-operativity, with Hill coefficients (h 1.6-1.9 at pH8.5), which are dependent on both pH and temperature. The highest co-operativity was observed at 20 degrees C and pH9 for haemoglobins I and II, whereas it was at 27 degrees C and pH8.5 for haemoglobin III. Thus the oxygen-binding behaviour of haemoglobin III in vitro is significantly different from those of haemoglobins I and II and indicates possibly its specific physiological role in vivo in the adaptive process in the natural environment.

Project description:We have cloned and characterized Xlrbpa, a double-stranded RNA-binding protein from Xenopus laevis. Xlrbpa is a protein of 33 kD and contains three tandemly arranged, double-stranded RNA-binding domains (dsRBDs) that bind exclusively to double-stranded RNA in vitro, but fail to bind either single-stranded RNA or DNA. Sequence data and the overall organization of the protein suggest that Xlrbpa is the Xenopus homologue of human TAR-RNA binding protein (TRBP), a protein isolated by its ability to bind to human immunodeficiency virus (HIV) TAR-RNA. In transfection assays, TRBP has also been shown to inhibit the interferon-induced protein kinase PKR possibly by direct physical interaction. To determine the function of Xlrbpa and its human homologue we studied the expression and intracellular distribution of the two proteins. Xlrbpa is ubiquitously expressed with marked quantitative differences amongst all tissues. Xlrbpa and human TRBP can be detected in the cytoplasm and nucleus by immunofluorescence staining and Western blotting. Sedimentation gradient analyses and immunoprecipitation experiments suggest an association of cytoplasmic Xlrbpa with ribosomes. In contrast, a control construct containing two dsRBDs fails to associate with ribosomes in microinjected Xenopus oocytes. Nuclear staining of Xenopus lampbrush chromosome preparations showed the association of the protein with nucleoli, again indicating an association of the protein with ribosomal RNAs. Additionally, Xlrbpa could be located on lampbrush chromosomes and in snurposomes. Immunoprecipitations of nuclear extracts demonstrated the presence of the protein in heterogeneous nuclear (hn) RNP particles, but not in small nuclear RNPs, explaining the chromosomal localization of the protein. It thus appears that Xlrbpa is a general double-stranded RNA-binding protein which is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs either alone or as part of an hnRNP complex.

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Effects of antibody to 5 S-RNA-binding protein on protein synthesis in Artemia salina ribosomes.

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