Biomedical subjects
Publications and source records attributed to H Bielka.
The decrease of the growth rate of the Ehrlich ascites tumor is correlated with changes in the de novo synthesis and abundance of several proteins as is shown by electrophoretic methods. The synthesis of a number of these growth-dependent proteins can be induced in vitro by incubation of the tumor cells in the presence of serum.
1. Two of the three isoforms of the growth-related protein p25 of the Ehrlich ascites tumor have been purified to homogeneity by giant two-dimensional polyacrylamide gel electrophoresis. 2. Antibodies raised against the isoform p25/1 react also with isoforms p25/2 and p25/3. 3. Limited tryptic digestion of p25/1 and p25/2 resulted in similar oligopeptide patterns. Corresponding oligopeptides of both isoforms have identical amino acid sequences. 4. The isoforms p25/2 and p25/3 are phosphorylated derivatives of unphosphorylated p25/1. The phosphorus is bound to serine and a further unknown phosphorylation site.
The decrease of the in vivo growth rate of the Ehrlich ascites tumor after intraperitoneal injection of carcinoma cells into mice is correlated with (1) a decline in the overall rate of cellular protein synthesis, (2) a corresponding decrease of the activity of the cytosolic fraction to stimulate in vitro polysomal protein synthesis, (3) the abundance and phosphorylation of a 25 kDa protein. The results are discussed with respect to correlations between the regulation of protein synthesis and cell proliferation.
Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and--to a lesser extent--L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.
The distribution of initiation factor 2(eIF-2) and elongation factor 2(EF-2) in cultured mouse embryo fibroblasts was studied and compared with the distribution of ribosomes. We used immunofluorescence microscopy with monospecific antibodies to eIF-2, EF-2, and proteins S3a and S7 of the small ribosomal subunit. Ribosomes and factors eIF-2 and EF-2 were found mainly in the vicinity of the cell nucleus. This perinuclear zone coincides with the endoplasm - the central part of the cell containing numerous membraneous organelles and inclusions. Besides the perinuclear zone, small stained regions could be seen at the periphery of some cells. After treatment of the cells with Triton X-100 in a buffer conditions, that stabilizes the major cytoskeletal structures, some of the ribosomes, eIF-2, and EF-2 remained bound to the insoluble material. These components were found near the nucleus and some were located along the microfilament bundles.
We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.
The protein synthesis-stimulating activity of the cytosolic fraction from regenerating rat liver was tested in a cell-free system using washed polysomes from normal rat liver. This activity undergoes significant changes during liver regeneration after partial hepatectomy (p.h.). An initial decrease until 16 h after p.h. is followed by a significant increase until 24 h after p.h. Beyond 32 h after p.h. the activity begins to decline again. Evidence is presented that these changes of the cytosolic activity may not be due to alterations in the distribution of protein synthesis-stimulating factors between the microsomal and the cytosolic fraction. The Met-tRNAf-binding activity of the cytosolic fraction changes during liver regeneration analogously to the protein synthesis-stimulating activity measured in the polysomal assay. This indicates that initiation factor eIF-2 is involved in the observed changes of the cytosolic activity. This conclusion could be confirmed by addition of purified eIF-2 to the polysomal assay system. Addition of eIF-2 to cytosolic fractions of low endogenous protein synthesis-stimulating activity (16 h after p.h.) enhances amino-acid incorporation to a significantly higher extent than addition to highly active cytosolic fractions (24 h and 32 h after p.h.). From these results it is concluded that changes in eIF-2 plays an essential role in the described alterations of the cytosolic activities during liver regeneration.
On the basis of hydrodynamic, electron microscopic and biochemical investigations a new model of the structure of initiation factor eIF-3 is proposed. From sedimentation and diffusion coefficients of 16.35 S and 2.13 X 10(-7) cm2/s, respectively, as well as from sedimentation equilibrium measurements, a molecular mass of about 650 kDa was determined for isolated eIF-3. This is in agreement with molecular mass estimations by sodium dodecyl sulphate gel electrophoresis. A partial specific volume of 0.723 cm3/g was determined by means of the amino acid composition and the specific volume increments of the amino acids. From this value and from the molecular mass, a volume of 780 nm3 was calculated for eIF-3. In electron micrographs of isolated eIF-3, images with triangular profiles and side lengths of 14 nm, 16 nm, and 17 nm have been observed. Taking into account the calculated volume and considering the triangular image as one face of the particle, it is suggested that eIF-3 has the shape of a flat triangular prism with a height of about 7 nm and the above-mentioned side-lengths. This model is in agreement with results of electron microscopic investigations of eIF-3 in native small ribosomal subunits [Lutsch, G., Benndorf, R., Westermann, P., Bommer, U.-A. & Bielka, H. (1986) Eur. J. Cell Biol. 40/2, in press]. The high frictional ratio of about 1.7 also supports eIF-3 to be rather of a flat than of a globular shape.
The interaction of rat liver ribosomes with poly(U), spin labeled (SL) at the 2'OH groups of ribose residues by N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole, has been studied by electron spin resonance (ESR) spectroscopy. The ESR spectra demonstrate that SL-poly(U) with a modification of 1 spin label per 20 ribose residues binds to 80S ribosomes as well as to 40S subunits in a 1:1 stoichiometry at 12 mM MgCl2. The same result is found with highly modified poly(U) bearing 1 SL per 4 ribose residues. Addition of excessive amounts of unmodified poly(U) displaces bound SL-poly(U) from the ribosome which points to a competition for the same binding site at the ribosome. The biological activity of SL-poly(U) was tested with regard to trigger 80S ribosomes for binding of Phe-tRNAPhe and for poly(Phe) synthesis. SL-poly(U) bearing 1 SL group per 20 ribose residues directs the binding of only 50% of the amount of Phe-tRNAPhe bound to ribosomes in the presence of unmodified poly(U). When SL-poly(U) bearing 1 SL group per 4 ribose residues is used, this value drops to 25%. Poly(Phe) synthesis is even more impaired: In the presence of poly(U) bearing 1 SL-group per 20 ribose residues only about 30% of the amount of poly(Phe) coded by unmodified poly(U) are synthesized and in the presence of SL-poly(U) bearing 1 SL group per 4 ribose residues poly(Phe) synthesis is completely abolished. The results suggest that modification of the ribose moiety has only a relatively small influence on the binding of mRNA to ribosomes but causes substantial impairment of the mRNA function, whereas, as shown earlier (Ebert et al., Acta Biol. Med. Germ. 41, 431, 1982), modification of the base moiety of poly(U) (in a proportion of 1 SL per 30 bases) does not influence coding efficiency for poly(Phe) synthesis.
The cytosolic fraction prepared from in vivo stationary phase Ehrlich ascites carcinoma cells (EAC cells) stimulates in vitro protein synthesis by isolated polysomes to a substantially lower extent than the cytosol of exponentially growing cells. The cytosolic fraction of EAC cells treated in vitro for 24 h with a purified 13 kD growth inhibitor from bovine mammary gland was by 20-40% less active in stimulating in vitro protein synthesis in comparison to control cytosols. It could be shown that the growth inhibitor does not act directly on the cytosol but rather exerts its action by (a) plasma membrane mediated mechanism(s). The results are discussed with respect to correlations between regulations of cell proliferation and protein synthesis at the posttranscriptional level.
Native small ribosomal subunits (40SN) from rat liver and rabbit reticulocytes prepared at different KC1 concentrations have been investigated by electron microscopy after negative staining. Subunits of both origins show identical features. The initiation factor eIF-3 is located in the middle region of the convex rear side of the particles and covers an area extending from the protuberance at the interface up to the external surface. eIF-3 has the shape of a flat triangular prism and is attached with its triangular base to the ribosomal surface.
Protein-RNA interactions in the 5S rRNA-protein L5 complex from rat liver ribosomes were studied by limited digestion of free and protein bound 5S rRNA with ribonuclease A and T1. In the complex with protein L5 the digestion of 5S rRNA by ribonuclease T1 is decreased at G37 and G89, whereas U38 and C39, and to a lower extent also C10 and U12 become accessible for ribonuclease A.
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The three-dimensional structure of initiation factor eIF-3 and its binding site on native small ribosomal subunits have been analyzed by electron microscopic studies of native small ribosomal subunits and of initiation factor eIF-3 prepared from rat liver as well as by hydrodynamic studies of isolated eIF-3. Initiation factor eIF-3 has the shape of a flat triangular prism and is bound with its triangular base to the body part of the convex rear side of the small ribosomal subunit.
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.
The location of ribosomal proteins S3a, S5, S6, S7 and S9 on the surface of 40S subunits of rat liver ribosomes has been determined by means of antibody labeling and electron microscopy. Multiple antigenic determinants could be detected for proteins S3a, S6 and S7, whereas protein S9 shows only one and protein S5 two closely neighboured antigenic determinants. Antigenic determinants of proteins S3a, S6 and S7 have been mapped in the head and the body region of the 40S subunits. Protein S5 could be localized in the head and protein S9 in the body of the 40S subunits. These findings are discussed with regard to the location of functional sites on the 40S ribosomal subunit.
This review deals with results of affinity labelling experiments on proteins of the peptidyl-transferase centre (PTC) of animal ribosomes. From the studies it is concluded that ribosomal proteins L 5, L 21/23, L 32/33, and L 36 are the main candidates for the ribosomal P site and proteins L 6, L 18a, and L 28/29 for the ribosomal A site. Proteins L 10 and L 13/15 seem to be located in a more central region of the PTC. These findings are correlated with results obtained so far by using other techniques. Finally, the data are discussed, and a tentative model on the relative spatial arrangement of certain ribosomal proteins at the PTC is proposed.
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