Biomedical subjects

D Elson

Publications and source records attributed to D Elson.

At least 37 records · Page 2Linked to original sources

PubMed1979

Interaction between the hepatic growth hormone receptor and concanavalin A.

The interaction between the plant lectin concanavalin A (Con A) and hepatic receptors for human growth hormone (GH) has been studied in particulate and soluble microsomal membrane preparations from rabbit and rat liver. Con A shows a dose-dependent, partial (30%) inhibition of 125I-human GH binding which is reversed by the Con A competitor, alpha-methyl mannoside. The Con A effect is dependent on the receptor concentration. The inhibition by Con A in rabbit liver is a reflection of a decreased number of available binding sites--there is no effect on binding affinity. It would appear that Con A binds directly to the GH-binding protein and not to an adjacent membrane glycoprotein. The GH receptor may consist of more than one molecular species, differing only in the carbohydrate type or content.

Animals↗

PubMed1978

A large nucleoprotein fragment of the 50-S ribosomal subunit of Escherichia coli.

A large nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 50-S ribosomes and purified to gel electrophoretic homogeneity. Conditions were employed under which the fragmentation pattern was reproducible and the various fragment fractions were stable and maintained their sedimentation and electrophoretic properties throughout the several preparative and analytical procedures used. Fractions that appeared homogeneous in sucrose gradient centrifugation were found to be heterogeneous by gel electrophoresis. The large fragment was purified to homogeneity by preparative gel electrophoresis. It contained 21 proteins, the 5-S RNA, and two large oligonucleotides which together total about two thirds the molecular weight of the 23-S RNA. Because it can be prepared reproducibly in large quantities and because of its size and stability, the fragment appears suitable for functional and structural studies and as the starting material for further fractionation. An important contributing factor to the observed stability and reproducibility was the maintenance of an unchanging ionic environment. A single buffer was employed throughout all the procedures, and the fragments produced by nuclease digestion were dissociated from each other by heat rather than by changing the medium.

Enzymes, Immobilized↗

PubMed1978

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.

Affinity Labels↗

PubMed1976

Conformational changes of 30S ribosomes measured by intrinsic and extrinsic fluorescence.

The intrinsic tryptophan fluorescence and the fluorescence of N-(3-pyrene)maleimide, a covalently bound sulfhydryl-specific extrinsic probe, have been used to study the conformation of the 30S ribosomal subunit of Escherichia coli. (a) The tryptophan fluorescenct spectrum of the free ribosomal proteins is shifted to shorter wavelengths than that of free tryptophan. When the proteins are incorporated into the organized structure of the ribosome, there is a small additional blue shift and the emission band becomes narrower. In 6 M urea, the spectrum of the proteins, whether free or in the ribosome, becomes identical with that of the amino acid, reflecting exposure of previously shielded tryptophan residues. (b) When magnesium-depleted ribosomes are unfolded at low ionic strength, the tryptophan fluorescence spectrum changes, although circular dichroism shows no change in alpha-helix content of the proteins. (c) Intrinsic and extrinsic fluorescence were both found to be sensitive to a limited and fully reversible transition that takes place when ribosomes are incubated under conditions that increase their activity in vitro. This suggests that both probes may be of use in monitoring conformational changes that occur under conditions consistent with activity. The kinetics of the concurrent changes in extrinsic fluorescence and aminoacyl-tRNA binding activity were compared. (d) Conditions are described for labeling ribosomes with N-(3-pyrene)maleimide without impairing their activity.

Circular Dichroism↗

PubMed1976

Synthesis of a cleavable protein-crosslinking reagent for the investigation of ribosome structure.

This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents, which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2-mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega-activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3'-dithiobispropionimidate, dimethyl 4,4'-dithiobisbutyrimidate, and dimethyl 6-6'-dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega-bromonitriles. Experiments with the bispropionimidate and a test protein, pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can recovered after treatment with 2-mercaptoethanol. The reagents are suitable for studies of ribosomal structure.

Binding Sites↗

PubMed1976

The use of a cleavable crosslinking reagent to identify neighboring proteins in the 30-S ribosomal subunit of Escherichia coli.

A cleavable bifunctional reagent, dimethyl 3,3'-dithiobispropionimidate, has been used to crosslink proteins that occupy neighboring positions in the 30-S ribosomal subunit of Escherichia coli. The crosslinked proteins were identified, fully or partly, by their positions in two two-dimensional gel electrophoretic systems, one diagonal and the other quasi-diagonal, in which the complexes were cleaved after the first-dimensional run. It was found to be necessary to block the protein sulfhydryl groups in order to prevent artifactual disulfide crosslinking after extraction of the protein from ribosome. Eleven crosslinked complexes were detected. Four were fully identified: the triplet S4-S5-S8, and the pairs S2-S3, S4-S5, and S5-S8. In five others one component was identified unambiguously. No additional complexes were seen when the longer homologous butyro and capro reagents were used.

Binding Sites↗

PubMed1976