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P Westermann

Publications and source records attributed to P Westermann.

At least 55 records · Page 3Linked to original sources
PubMed1984

Cross-linking of mRNA to initiation factor eIF-3, 24 kDa cap binding protein and ribosomal proteins S1, S3/3a, S6 and S11 within the 48S pre-initiation complex.

Native small ribosomal subunits from rabbit reticulocytes contain all initiation factors necessary for the formation of the mRNA-containing 48S pre-initiation complex. The complex formed in the presence of Met-tRNAf and 125I-labelled globin mRNA was cross-linked with diepoxybutane, and the covalent mRNA-protein complexes were isolated under denaturating conditions. The proteins of the covalent complex were identified as the 110, 95 and 66/64 kDa subunits of eIF-3. In addition, the 24 kDa cap binding protein and the ribosomal proteins S1, S3/3a, S6 and S11 were found covalently linked to the mRNA. Ribosomal proteins S3/3a and S6 were also involved in the ribosomal mRNA-binding domain of reticulocyte polysomes.

Animals↗
PubMed1983

The spatial arrangement of the complex between eukaryotic initiation factor eIF-3 and 40 S ribosomal subunit. Cross-linking between factor and ribosomal proteins.

The binding site for eIF-3 on the small ribosomal subunit was studied (a) by use of a complex of eIF-3 and derived 40 S ribosomal subunit from rat liver, and (b) by use of native small ribosomal subunits from rabbit reticulocytes. After treatment of both complexes with dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate ribosomal proteins S3a, S4, S6, S7, S8, S9, S10, S23/24 and S27 became covalently linked to eIF-3 and were isolated together with the factor by gradient centrifugation. The ribosomal proteins were identified by two-dimensional polyacrylamide gel electrophoresis after periodate cleavage of the link(s).

Animals↗
PubMed1983

Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.

Cross-linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate produced numerous covalently linked protein dimers which could be separated by a combination of ion-exchange chromatography on carboxymethyl cellulose and polyacrylamide gel electrophoresis. The protein components of the dimers were identified electrophoretically after periodate cleavage of the cross-link(s). The analysis revealed 42 cross-linked dimers involving 25 different proteins. Among these, proteins S3, S4 and S20 occurred in combinations with six, eight and seven different proteins, respectively. For proteins S13, S14 and S17 five protein neighbours could be identified, while 13 of the remaining proteins were linked to three or four different protein partners. The involvement of the majority of proteins in the formation of multiple cross-linked dimers implies that a large number of protein-protein interaction sites exist within the ribosomal subunit. A preliminary model illustrating the arrangement of 16 proteins in the small ribosomal subunit is presented and discussed with respect to possible functions, especially in the event of translation initiation.

Animals↗
PubMed1983

Chemical crosslinking of major structural proteins within type D retroviruses.

The nearest neighbor relationships of major structural proteins within type D retroviruses (SMRV, MPMV, PMFV) were investigated by crosslinking with the cleavable bifunctional reagent dimethyl dithiobispropionimidate (DTBPI) or by use of 2-iminothiolane (methyl mercaptobutyrimidate, MBI) and subsequent oxidation by hydrogen peroxide. The crosslinked complexes of proteins were analyzed by two-dimensional diagonal polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In intact virions both crosslinking reagents induced the formation of covalently linked complexes of the internal structural proteins as well as complexes containing the envelope glycoproteins. The following complexes were found for each virus: SMRV: (pp20)2; (p35)2,4,6; (pp20-gp68); MPMV: (p10)2,4,6; (p25)2,4; (p10-p25); (gp20-gp68); (p45-gp68); PMFV: (p10)2; (p25)2; (p10-p25); (p45-gp68). In control experiments without crosslinking reagents no covalently linked complexes were detected.

Cross-Linking Reagents↗
PubMed1982

Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose.

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25-40% saturation (0 degree C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation, eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3, sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.

Animals↗
PubMed1982

Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3 small ribosomal subunit, as shown by cross-linking experiments.

Initiation factor eIF-3 from rat liver forms a binary complex with the small ribosomal subunit. Within this complex, 18S ribosomal RNA can be cross-linked to the 66 000 dalton subunit of eIF-3 by treating the complex with a short bifunctional reagent, diepoxybutane, with a distance of 4A between the reactive groups. In binary complexes containing eIF-3 premodified with the heterobifunctional reagent, methyl-p-azido-benzoylaminoacetimidate (10A), the 66 000 dalton subunit of eIF-3 became covalently bound to 18S rRNA after irradiation of the complex with ultraviolet light. The involvement of only one of the eight eIF-3 subunits in the formation of the covalent RNA-protein complexes indicates a highly specific interaction between 18S rRNA and eIF-3 at the attachment site of the factor on the 40S subunit.

Animals↗
PubMed1981

Cross-linking of Met-tRNAf to eIF-2 beta and to the ribosomal proteins S3a and S6 within the eukaryotic inhibition complex, eIF-2 .GMPPCP.Met-tRNAf.small ribosomal subunit.

In the quaternary initiation complex, eIF-2.GMPPCP.Met-tRNAf.40S ribosomal subunit, the Met-tRNAf can be cross-linked to the beta subunit of initiation factor eIF-2 as well as to ribosomal proteins S3a and S6 by treatment with the bifunctional reagent, diepoxybutane. Using 40S subunits, modified in advance with the heterobifunctional reagent, methyl-rho-azido-benzoylaminoacetimidate, Met-tRNAf is covalently bound to the same ribosomal proteins (S3a and S6) upon irradiation of the complex with ultraviolet light. Under both conditions proteins S3a and S6, together with a limited number of other ribosomal proteins, are covalently bound to 18S ribosomal RNA.

Cross-Linking Reagents↗
PubMed1980

The alpha and gamma subunits of initiation factor eIF-2 can be cross-linked to 18S ribosomal RNA within the quaternary initiation complex, eIF-2.Met-tRNAf.GDPCP.small ribosomal subunit.

Initiation factor eIF-2 from rat liver was reacted with the hetero-bifunctional cross-linking reagents ABAI or APTPI without diminishing its ability to form the quaternary initiation complex with Met-tRNAf, GDPCP and the small ribosomal subunit. Upon irradiation with UV light, subunits alpha and gamma of eIF-2 became covalently linked to 18S ribosomal RNA. The subunits were identified electrophoretically after isolation of the covalent protein-rRNA complexes and subsequent degradation of the rRNA by nuclease and alkali treatments. The close proximity of the two factor subunits to sequences of ribosomal RNA within the quaternary complex could be confirmed in a second set of experiments using unmodified, 125I-labeled factor and diepoxybutane as cross-linking reagent.

Animals↗
PubMed1980

The use of rRNA-cellulose chromatography in the rapid isolation of homogeneous protein synthesis initiation factor eIF-2 from rat liver microsomes.

A rapid and efficient procedure is described for preparing homogeneous polypeptide chain initiation factor eIF-2 from rat liver microsomal salt wash. The method involves ammonium sulfate fractionation, affinity chromatography on rRNA-cellulose and DEAE-cellulose chromatography. The preparation is highly active in initiation complex formation.

Animals↗
PubMed1980

Neighbourhoods of proteins S2--S3, S3--S3a, S15--S15a, and S5--S25 within the small subunit of rat liver ribosomes.

Cross-linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 3.8-diaza-4.7-dioxo-5.6-dihydroxydecanbisimidate results in the formation of various complex which were separated by two-dimensional polyacrylamide gel electrophoresis. Components of four complexes were electrophoretically identified after periodate cleavage as S2--S3, S3--S3a, S15--S15a, and S5--S25.

Animals↗