The attachment of polyuridylic acid to reticulocyte ribosomes

The attachment of polyuridylic acid to reticulocyte ribosomes was studied by using polyadenylic acid, which inhibits the attachment reaction only, while permitting translation of polyuridylic acid bound to ribosomes. After addition of polyadenylic acid the amount of polyphenylalanine synthesized under standard conditions was taken as a measure of the bound polyuridylic acid. In this way certain parameters of the attachment reaction and the subsequent translation of attached polyuridylic acid were defined: (1) polyuridylic acid-ribosome interaction at 37 degrees requires only Mg(2+) at an optimum concentration of 8mm; (2) K(+) (required for translation) is a non-competitive inhibitor of the attachment reaction; (3) optimum polyphenylalanine synthesis directed by attached polyuridylic acid occurs at 5mm-Mg(2+) concentration; (4) from kinetic studies single ribosomes appear to participate in the attachment reaction.     

The occurrence of gamma-carboxyglutamic acid in mammalian ribosomes.

γ-carboxyglutamic acid (GLA) has been identified and estimated in mammalian ribosomes (mouse, rat, man, cultured cells) by amino acid analysis of total extracted protein. Assay results varied from 5.7–19.1 nMoles GLA/mg protein (6.0–9.9 residues GLA/1000 residues GLU), depending on purity and origin of ribosomes. GLA was not eliminated by purification. GLA in mouse hepatoma ribosomes was increased 3-fold upon preparation of puromycin-dissociated sub-units and sedimentation on gradients containing 1.0 M KCl. This tightly bound GLA (17.7 nMoles/mg sub-unit protein) could be present in one or more ribosomal proteins incorporated in the nucleoli, or in GLA-containing proteins acquired in the cytoplasm.     

Proteins of mammalian mitochondrial ribosomes.

The bovine mitochondrial system is being developed as a model system for studies on mammalian mitochondrial ribosomes. Information is emerging on the structural organization and RNA binding properties of proteins in these mitochondrial ribosomes. Unexpectedly, these ribosomes appear to interact directly with GTP, via a high affinity binding site on the small subunit. Despite major differences in their RNA content and physical properties, mammalian mitochondrial and cytoplasmic ribosomes contain about the same number of proteins. The proteins in each kind of ribosome have a similar size distribution, and both sets are entirely coded by nuclear genes, raising the possibility that these different ribosomes may contain the same set of proteins. Comparison of bovine mitochondrial and cytoplasmic r-proteins by co-electrophoresis in two-dimensional gels reveals that most of the cytoplasmic ribosomal proteins are more basic than the mitochondrial ribosomal proteins, and that none are co-migratory with mitochondrial ribosomal proteins, suggesting that the proteins in the two ribosomes are different. To exclude the possibility that the electrophoretic differences result only from post-translational modification of otherwise identical proteins, antibodies against several proteins from the large subunit of bovine mitochondrial ribosomes were tested against cytoplasmic ribosomes by solid phase radioimmunoassay and against cytoplasmic ribosomal proteins on Western blots. The lack of cross-reaction of these antibodies with cytoplasmic r-proteins suggests that mitochondrial ribosomal proteins have different primary structures and thus are most likely encoded by a separate set of nuclear genes.     

Affinity labeling of a reactive sulfhydryl residue at the peptidyl transferase P site in Drosophila ribosomes.

An affinity label has been prepared that is specific for the P site of a eucaryotic peptidyl transferase, that of Drosophila melanogaster. It has the sequence C-A-C-C-A-(Ac[3H]Leu) with a mercury atom added at the C-5 position of all three cytosine residues (referred to as the mercurated fragment). This label is an analogue of the 3' terminus of N-acetylleucyl-tRNA. The mercurated fragment binds specifically to the P site of peptidyl transferase. It participates fully in peptide bond formation as judged by its ability to transfer N-acetylleucine to puromycin with at least the same efficiency as a nonmercurated fragment. Once bound to the P site, the mercurated fragment reacts covalently with a ribosomal protein(s). This affinity-labeling process can be effectively competed by nonmercurated fragment, which indicates a site-specific reaction. The covalent attachment of the affinity label to a ribosomal protein(s) occurs through the formation of a mercury-sulfur bond, as judged by its lability in the presence of thiol reducing agents. The major ribosomal protein labeled at the P site of D. melanogaster was found to be a small, basic protein. The electrophoretic behavior of this protein parallels that of major P site proteins found in Escherichia coli ribosomes and in other eucaryotes. These results suggest conservation of some of the overall properties of the P site proteins from these organisms.     

The attachment of UDP-hexosamines to the ribosomes isolated from rat liver

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.     

Yeast ribosomes bind to highly purified reconstituted Sec61p complex and to mammalian p180

To determine whether the yeast Sec61p translocation pore is a high-affinity ribosome receptor in the endoplasmic reticulum, we isolated the Sec61p complex using an improved protocol in which contaminants found previously to be associated with the complex are absent. The purified complex, which contains Sec61p with an amino terminal hexahistidine tag, was active since it rescued a sec61–3 post-translational translocation defect in a reconstituted system. Co-reconstitution of the Sec61p and Sec63p complexes into liposomes failed to support post-translational translocation, suggesting that Sec62p is required for this process. By Scatchard analysis, the purified Sec61p complex bound to yeast ribosomes when reconstituted into liposomes with a K_D of 5.6 n m , a value similar to the K_D obtained when ribosome binding to total microsomal protein was measured (2.7 n m ). In addition, a mammalian protein, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this strain exhibited ∼2.3-fold greater binding to yeast ribosomes. Despite this increase in ribosome binding, neither co- nor post-translational translocation was compromised in vivo . In sum, our data suggest that the Sec61p complex is a ribosome receptor in the yeast endoplasmic reticulum membrane.     

The nature of the multiple forms of D-erythrodihydroneopterin triphosphate synthetase.

1. Three forms of the Lactobacillus plantarum enzyme D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis, have been demonstrated by polyacrylamide gel electrophoresis. The enzyme forms designated the alpha prime, alpha and beta forms have been shown to be conformers with molecular weights of approx. 200 000. Study of the subunit structure of the beta enzyme species by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a single protein with an estimated molecular weight of 20 000 which suggests that the enzyme molecule may be composed of ten polypeptide chains. 2. Of the three conformers only one form, the beta form, appears to be enzymatically active. The two other conformers must undergo conformational changes to the beta species before enzymatic activity can be demonstrated in reaction mixtures containing these enzyme forms. 3. The three enzyme species are interconvertible. The removal of phosphate ions from the enzymatically active beta form results in the formation of two inactive species which suggests that the conformation of the active enzyme is stabilized by non-covalently bound phosphate ions. Conversion of the inactive species to the beta enzyme form may be effected by the readdition of phosphate, substrate or certain nucleotides.     

Specificity of attachment of certain enterobacteriaceae to mammalian cells.

The specificity of adherence of various Enterobacteriaceae to different mammalian cells was studied in vitro. 3H-Labelled organisms of the same species isolated from various clinical sources differed significantly in their abilities to adhere to the same mammalian cells. Bacteria frequently adhered better to cells derived from sites other than those analogous to their original source. Bacteria did not display consistently 'high' or 'low' attachment to a variety of human and tissue-cultured cells and little selective adherence was demonstrable.     

Organization of proteins in mammalian mitochondrial ribosomes. Accessibility to lactoperoxidase-catalyzed radioiodination

To assess the relative exposure of individual ribosomal proteins (r-proteins) in the large and small subunits of the bovine mitochondrial ribosome, we used a double label iodination technique. Regions of r-proteins exposed in purified ribosomal subunits were labeled with 131I using the lactoperoxidase-catalyzed iodination system, and additional reactive groups available upon denaturing the r-proteins in urea were labeled with 125I using the chloramine-T mediated reaction. The ratio of 131I to 125I incorporated into individual proteins under these conditions is representative of the degree of exposure for each of the proteins in the subunits. In this manner, the r-proteins have been grouped into 3 classes depending on their degree of exposure: high exposure, intermediate exposure, and essentially buried. While both subunits have a few proteins in the "highly exposed" group, and a large number of proteins in the "intermediate exposure" group, only the large ribosomal subunit has an appreciable number of proteins which appear essentially buried. The more buried proteins may serve mainly structural roles, perhaps acting as "assembly proteins," since many from this group bind to ribosomal RNA. The more superficially disposed proteins may comprise binding sites for macromolecules that interact with ribosomes during protein synthesis, as well as stabilizing the association of the large and small subribosomal particles.