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Biomedical subjects

H Bielka

Publications and source records attributed to H Bielka.

At least 73 records · Page 4Linked to original sources
PubMed1979

Photoaffinity labeling of rat liver ribosomes by N-(2-nitro-4-azidobenzoyl)puromycin.

N-(2-Nitro-4-azidobenzoyl)-[3H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes.

Affinity Labels↗
PubMed1978

Studies on the structure of rat-liver polysomes by small-angle X-ray scattering.

The spatial structure of rat liver polysomes in dilute solution has been studied by the small-angle X-ray scattering technique. The distance between the mass centres of neighbouring ribosomes on the mRNA molecule is 35 nm, and the distance from next-but-one neighbour, about 55 nm. The spatial arrangement of the ribosomes on the polysome is compact; stretched configurations can be ruled out. The best model is a solenoid with six ribosomes per turn and a pitch of about 33 nm. The greatest diameter of a polysome with eight ribosomes is 130 nm, the radius of gyration 34 nm.

Animals↗
PubMed1978

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions.

1. By enlarging the dimensions of the gels used in the usual analytical two-dimensional polyacrylamide gel electrophoresis it is possible to separate much larger amounts of ribosomal protein in comparison to analytical separations. 15 mg of protein mixture of small or large subunits of rat liver ribosomes can be separated by this procedure. 2. The positions of the proteins in the two-dimensional patterns are identified with a special staining procedure. The proteins are eluted from the gels with SDS/phosphate buffers. 3. The purity of the extracted proteins was tested by one-dimensional SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis, respectively. 24 proteins of the small and 24 proteins of the large ribosomal subunit were isolated in pure form. 4. The amino acid compositions of 24 proteins of the small and of 19 proteins of the large subunit were determined.

Amino Acids↗
PubMed1978

Number and molecular weights of the basic proteins of rat liver ribosomes.

1) Efficient separation of the proteins from rat liver ribosomes can by achieved by two-dimensional polyacrylamide gel electrophoresis. Complete separation of all components, however, is not possible with one system only. Comparison of the results obtained with different systems suggests further heterogeneity of S15, L22, L28, L33 and L35 and enables identification of S15a, S15b, L22a, L22b, L28a, L28b, L33, L33a, L35a and L36b. 2) Ribosomal proteins were substituted with iodoacetamide prior to electrophoresis or handled in all steps of the procedure in the presence of reducing agents. These procedures prevent the formation of oxidation products described erroneously as ribosomal proteins S5, S6, L15, L17 and L32 in earlier papers. 3) Estimation of the molecular weights was performed by two-dimensional separation of the small and large subunit proteins using sodium dodecyl sulphate in the second dimension. The positions of the 70 basic proteins in the 2-D patterns were identified. 4) The small and large subunit proteins have molecular weights in the range of 8000 to 35,000 and 11,000 to 55,500 Dalton, respectively. The number average molecular weights for the small and large subunit proteins are 22,500 and 26,500 Dalton, respectively. The sum of the molecular weights is 0.67 x 10(6) Dalton for the proteins of the small subunit and 1.05 x 10(6) Dalton for the proteins of the large subunit.

Acetates↗
PubMed1978

Preparation and properties of a Met-tRNAf binding factor from rat liver and rat hepatoma.

A Met-tRNAf binding factor (IF-2) from the microsomal fraction of rat liver and rat hepatoma ascites cells was partially purified by ammonium sulphate fractionation, DEAE-cellulose and phosphocellulose chromatography. The factor binds [3H]Met-tRNAf only in the presence of either GTP or GMPPCP. Maximal binding takes place at 37 degrees C and in the absence of Mg++. The factor is specific for Met-tRNAf and does not bind Phe-tRNA from rat liver or from E. coli. The ternary complex [Met-tRNAf . IF-2 . GTP1 binds to 40 S ribosomal subunits from rat liver in the absence of mRNA or poly(A, G, U) without GTP hydrolysis. GDP as well as aurintricarboxylic acid inhibit the ternary complex formation. Both factors are rapidly inactivated by N-ethylmaleimide treatment and by preincubation at 45 degrees C. Heat inactivation is partially prevented by GTP and GDP. With regard to the functional properties there are no significant differences between IF-2 from normal liver and hepatoma cells. On the other hand heat denaturation compared to the rat liver factor, which may be due to differences in contaminating proteins.

Animals↗
PubMed1978

Studies on proteins of animal ribosomes. XXVIII. Preparation and antigenic properties of 40 S subunit proteins of rat liver ribosomes.

By combination of ion exchange chromatography, gel filtration, preparative polyacrylamide gel electrophoresis and perchloric acid fractionation 21 proteins of the small ribosomal subunit of rat liver were isolated with a purity of more than 95%. It could be demonstrated by immunological studies that no extensive structural homologies exist between these proteins.

Animals↗
PubMed1977

Presence of an endopeptidase activity in rat liver ribosomes.

Preparation of ribosomes using different procedures (treatment of postmitochondrial-postlysosomal supernatant or microsomes with 1% triton in 0.15 or 0.5 M KCl and subsequent sucrose gradient centrifugation; treatment of microsomes with 1.5% deoxycholate/2% triton) results in purified ribosomes which contain an endopeptidase activity detectable by breakdown of ribosomal proteins to trichloroacetic acid soluble split products. The proteolytic activity can be recovered also in the extracted proteins of whole ribosomes. With ribosomes the pH optimum of proteolytic breakdown is at about 7. The inhibition of the activity by leupeptin, DIFP and soya bean trypsin inhibitor suggests a serine type of the proteolytic activity.

Animals↗
PubMed1976

[Age dependent changes of biosynthesis of proteins (author's transl)].

The activity of KC1-washed ribosomes from the liver and the kidney of rats in a poly(U) dependent cell free system with [3H]-phe-tRNA as phenylalanine donor is highly increased by the cytosolic fraction from liver, brain, kidney and testicles. The activity of the cell sap of these tissues decreases significantly with increasing age of the rats from which the cytosolic fractions were prepared. Ribosomes do not undergo age dependent alterations with regard to their activity in poly(U) systems supported by cell sap from rat liver. The results are discussed with regard to age dependent change of the content of cytosolic translation factors.

Aging↗
PubMed1976

Ionic interactions in eukaryotic ribosomes: splitting of the subunits of rat liver ribosomes by treatment with monovalent cations.

Proteins of rat liver ribosomal subunits were successively split off by stepwise treatment of the particles with various concentrations of LiC1 (0.4--1.0 M) or KC1 (0.6--1.5 M). Split proteins and core particle proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The LiC1 concentrations were estimated at which the individual proteins are split off. The proteins of 40S and 60S subunits were classified in 4 and 5 groups, respectively, according to their splitting behavior. The effect of LiC1 and KC1 is similar, but higher concentrations of KC1 than of LiC1 are required to obtain comparable split protein patterns. Evidence for structural changes of the core particles during the split procedures was obtained.

Animals↗
PubMed1975

Changes in the reactivity of proteins of rat liver ribosomes against [14Ciodoacetamide depending on their organization in ribosomal subparticles, 80S ribosomes and on the attachment of poly-(U).

(1) The isolated mixtures of ribosomal proteins can be substituted by [14C]-iodoacetamide up to an average of about 2 equivalents per 20,000 dalton. The extent of substitution of single proteins measured after two-dimensional polyacrylamide gel electrophoresis shows that all proteins are reactive. (2) Also in the subunits, all proteins are accessible to substitution. Compared with isolated proteins, however, the reactivity is decreased and the amount of labelling for most proteins ranges as low as 5 to 20%. (3) Reassociation of ribosomal subunits decreases the reactivity of 12 proteins of the small subunit and that of 20 proteins of the large subunit. (4) The presence of messenger inhibits the substitution of 10 proteins of the small subunit and of 6 proteins of the large one. (5) Seven proteins of the small subunit and 3 proteins of the large one are influenced both by the other subunit and by messenger-RNA.

Animals↗
PubMed1975

[Structure of animal ribosomes. VI. Adynamic model of ribosome structure].

A dynamic model for the structure of ribosomes is developed using X-ray small-angle scattering data and electron micrographs of ribosomes and polysomes. The large subunit has the shape of a conical frustum, and has a groove at its flat side. The small subunit consists of two semiparticles which are connected by a molecular strand; it may assume two conformations: a prolate P conformation and an oblate O conformation. Translocation of the ribosome along the mRNA in the elongation process of protein synthesis is accomplished by cyclic conformation transitions P-O-P-O in combination with the linking and rupturing of bonds. This mechanism is called "rack and roll" mechanism.

Animals↗
PubMed1975

[Properties of ribosomes of normal and tumor cells (author's transl)].

On the basis of results showing that the extent and direction of protein biosynthesis essential for cell growth and cell differentiation can be regulated, aside from the transcription level, also on the level of ribosomes (translation), the main results of various properties of ribosomes from normal and tumor tissues are described. The main established difference between the two tissues concerns the organization and localization of the ribosomes within the cell. In tumors, polysomes and inactive dimers and monomeric particles are encountered in considerable amounts freely in the cytosol, i.e. not attached to the membranes. Furthermore, the polysomal profile of tumor cells compared with the normal tissue is characterized by a shift towards fractions with lower sedimentation coefficients. Monomeric ribosomes were not found to differ in physical and chemical properties. In smaller oligonucleotide fragments of ribosomal RNA, differences in the frequency of certain dinucleotides appear likely. Hybridization-competition experiments with DNA do suggest, however, that the ribosomal RNA from tumors is being synthesized at the same cistrons as ribosomal RNA from normal tissues, thus proving its overall sequence homology. Electrophoretic and immunologic methods have not so far revealed any clear differences in the properties of ribosomal proteins from normal and tumor tissues. In in-vitro systems, ribosomes from various tumors show approximately the same incorporation rates for amino acids as ribosomes from normal tissues. The enhanced protein synthesis of the cells, on the basis of data obtained from in-vitro experiments with cell free systems, is to be attributed mainly to the increased content of translation factors in the cytosol.

Animals↗
PubMed1975

Cycloheximide resistance in Chinese hamster cells. III. Characterization of cell-free protein synthesis by polysomes.

Two clones were selected for mass cultivation from 18 phenotypically stable CHM-resistant CHO clones. The polysomes isolated from these two clones were compared with CHO wildtype polysomes and rat liver polysomes in a cell-free protein synthesis system for their ability to incorporate amino acids. CHM had an inhibitory effect on the protein synthesis activity of CHO wildtype and rat liver polysomes, but had no effect on the polysomes obtained from either of the mutant CHO clones.

Amino Acids↗