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At least 19 recordsLinked to original sources

PubMed1979

The polysomal proteins of L cells. Discrimination between the structural ribosomal proteins, the exchangeable ribosomal proteins and the non-ribosomal proteins by two-dimensional dodecylsulfate electrophoresis and autoradiography.

Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two-dimensional polyacrylamide/dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non-radioactive spot in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and stained radioactive spots; (c) non-ribosomal proteins which are detectable only by autoradiography of gels. The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights less than or equal to 50 000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non-ribosomal proteins migrate in the region of the related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non-ribosomal proteins in polysomes are discussed.

Dactinomycin↗

PubMed1992

Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins.

The ets-related protein GABP alpha interacts with the four ankyrin-type (ANK) repeats of GABP beta to form a high-affinity DNA-binding complex that recognizes a site important for herpes simplex virus type I immediate early gene activation. To investigate the selectivity and specificity of the GABP complex, we have isolated two new ETS family members, termed ER81 and ER71. ER81 and GABP alpha were present in most tissues of adult mice, whereas ER71 was restricted to testis. We have compared the DNA-binding specificities of these proteins by binding site selection. GABP alpha, ER71, and ER81 recognized the common pentanucleotide DNA sequence 5'-CGGAA/T-3'. Although subtle differences were observed for nucleotide preferences flanking this pentanucleotide core, the overall similarity of the selected sequences was most striking. Given the observation that GABP alpha interaction with GABP beta requires its intact ETS domain, we further compared the ability of GABP beta to interact with other ETS proteins. GABP beta did not augment the DNA-binding activity of the highly similar ETS domains of ER81, ER71, or Ets-1. Moreover, probing of total tissue extracts with radiolabeled GABP beta demonstrated its exceedingly stringent specificity for GABP alpha. Given that the DNA-binding specificities of these ETS proteins are similar and that the protein-protein interactions between GABP beta and GABP alpha are highly specific, we conclude that the protein interactions determine the target site selection by GABP alpha.

Amino Acid Sequence↗

PubMed1976

Protein-protein interaction in transport: periplasmic histidine-binding protein J interacts with P protein.

A component of the high-affinity histidine transport system in Salmonella typhimurium, the periplasmic histidine-binding protein J, interacts with another transport component, the P protein. A mutant J protein, with a defective interaction site but intact histidine-binding site, can function in histidine transport if an appropriate compensating mutation is introduced in the P protein. The interaction between the J and P proteins is an obligatory step in transport. The significance of this interaction and of the involvement of the P protein in multiple transport functions is discussed.

Bacterial Proteins↗

PubMed1975

Protein-protein interactions of light-harvesting pigment protein from spinach chloroplasts. I.Ca-2+ binding and its relation to protein association.

The role of divalent cations in the regulation of the distribution of excitation energy between the two photosystems involved in green plants photosynthesis has led us to search for a better understanding of how such phenomena might occur at the molecular level. Since small changes in orientation of and distance between pigment molecules could greatly affect the distribution of excitation energy, we have decided to study the effects of ions on the light-harvesting pigment protein from spinach chloroplasts. The light-harvesting pigment protein is shown to have two types of binding sites for Ca-2+. Binding studies and analytical ultracentrifugation indicate that site I (K-d equals 2.5 mu-M, n equals 1.5-4.0 mu-mol Ca-2+ bound/mg chlorophyll) is lost as the protein associates. Site II (K-d equals 32 mu-M, n equals 9,5 mu-mol Ca-2+/mg chlorophyll) is not affected by the association of the protein. This site is responsible, however, for a further divalent cation-dependent association of the protein. The possible role of this protein in grana stacking and control of spillover is discussed.

Calcium↗

PubMed1976

[Protein utilization of mixed feed rations in lactating pigs with reference to the essential amino acid content of the feed proteins. 3. Report. Correlation between the protein metabolism data and the amino acid content of the dietary protein].

Protein utilization data were established in N balance trials with lactating sows fed various dietary proteins. The data obtained were compared with the amino acid indices of the dietary proteins and regression equations were established for the relations existing between these data. Additionally, simple and multiple regressions were calculated for relationships between the levels of N balance and the levels of crude protein intake, digestible crude protein, lysine per kg of milk production or lysine and all the other essential amino acids per 10 g of milk N. A relation was found between the level of daily lysine intake and all the other essential amino acids and the level of productive N utilization.

Amino Acids, Essential↗

PubMed1992

Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.

Animals↗

PubMed1979

Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.

Animals↗

PubMed1978

[Effect of the frequency of distribution of a protein supplement combined with a protein-free or low-protein diet on rat development. Nutritional effectiveness, body composition, liver nucleic acid and protein content, free plasma amino acids].

Irregular quantity and quality of food consumption is a common nutritional pattern in Marocco. Periods of liberal food supply due to a good harvest and festivals alternate with periods of more or less severe food shortage, resulting in a poor response to a high quality proteic diet taken from time to time. The object of this work was to study the effect of an irregular feeding sequence on the efficiency of food and the growth of the organism. Experiments on rats cannot be directly transposed to man, but are suggestive of the adverse effects of such a treatment. Discontinuity of protein intake leads to a decrease of its value as a complement for a poor basic diet, and this is more marked with the poorer diets and the longer intervals of complementation. Protein and energy retention decrease with the frequency of distribution of the protein supplement. Liver DNA increases when the supplementation is given with longer intervals; on the contrary the liver RNA decreases. The ratio RNA/DNA and proteins/DNA decreases still more. Blood amino-acids content depends on the composition of the diet and on the hormonal status of the organism.

Amino Acids↗

PubMed1975

The major proteins of the Escherichia coli outer cell envelope membrane. Characterization of proteins II* and III, comparison of all proteins.

Protein II*, one of the major Escherichia coli outer cell envelope membrane proteins has been characterized. The protein is heat-modifiable and perhaps due to complete unfolding and/or binding of sodium dodecylsulfate only at higher temperatures the modified protein exhibits a higher apparent molecular weight (33,000) than the non-modified form (28,000). Protein-chemical evidence as well as the behavior of two mutant proteins II* very strongly suggest that this protein consists of a single polypeptide chain and that in the strains studied there is no other major protein with similar characteristics. For another outer membrane protein, protein III (molecular weight 17,000), it has not yet been established if it should be classified as a major protein. Protein III consists of one or perhaps two polypeptide chains. The possibility existed that protein III is bound covalently to lipopolysaccharide, and this has been ruled out. Also, the lipopolysaccharide of the E. coli strains studied does not carry covalently bound protein in amounts anywhere near stoichiometry. N-on-protein substituents were neither found in protein II* nor in protein III. It is concluded that in E. coli B/r and the E. coli K12 strains used there are three major proteins: I, II, and IV; protein III may also belong to this class. There are not more major proteins than these. All four proteins are compared and discussed regarding their unknown functions and their relation to E. coli outer membrane proteins studied by other authors.

Amino Acids↗

PubMed1976

Isolation and characterization of hydrophobic proteins (H proteins) in the membrane fraction of Bacillus subtilis. Involvement in membrane biosynthesis and the formation of biochemically active membrane vesicles by combining H proteins with lipid.

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter. We named this H-protein. H-protein formed trilamellar unit membrane structure when combined with lipid. In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H-protein. However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein). Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid. H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid. Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems. Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.

Alanine↗

PubMed1979

Fractionation of two protein kinases from avian myeloblastosis virus and characterization of the protein kinase activity preferring basic phosphoacceptor proteins.

Two protein kinase activities were fractionated from purified virions of avian myeloblastosis virus. Distinguishing characteristics of these two protein kinases included: (i) their binding properties during purification by ion-exchange chromatography; (ii) their estimated molecular weights; and (iii) their phosphoacceptor protein specificities. The protein kinase that bound to the anion exchanger DEAE-cellulose (pH 7.2) had an estimated molecular weight of 60,000 to 64,000 and preferred basic phosphoacceptor proteins. The protein kinase that bound to the cation exchanger phosphocellulose (pH 7.2) had an estimated molecular weight of 42,000 to 46,000 and preferred acidic phosphoacceptor proteins. The protein kinase preferring basic phosphoacceptor proteins was further purified and characterized. Optimal transfer of phosphate catalyzed by this enzyme required a divalent metal ion, a sulfhydryl-reducing agent, and ATP as phosphate donor. GTP was not an effective phosphate donor at concentrations comparable to ATP; and the cyclic nucleotides cyclic AMP and cyclic GMP neither stimulated nor inhibited protein phosphorylation by the protein kinase. The specificity of the protein kinase for basic phosphoacceptor proteins extended to proteins from avian myeloblastosis virus, in that the neutral to basic virion proteins p12, p19, and p27 served as phosphate acceptors. In addition, the protein kinase also appeared to phosphorylate itself. The role(s) of this virion-associated protein kinase is discussed.

Avian Leukosis Virus↗

PubMed1977

Outer membrane proteins of Escherichia coli. V. Evidence that protein 1 and bacteriophage-directed protein 2 are different polypeptides.

Protein 1 from the outer membrane of Escherichia coli K-12 and protein 2 from a phage PA-2 lysogen of the same strain were isolated by differential sodium dodecyl sulfate extraction and purified by ion-exchange and gel filtration chromatography. Rabbit antisera were prepared against these proteins and showed no cross-reaction between proteins 1 and 2. The proteins have the same N-terminal amino acid but show small yet significant differences in amino acid composition. The proteins were cleaved with cyanogenbromide in solvents containing both formic acid and trifluoroacetic acid. By comparing the cleavage in these solvents, it was established that protein 1 yielded 5 cyanogen bromide peptides, and the sum of the molecular weights of these was equivalent to the molecular weight of the uncleaved protein. Protein 2 yielded 4 cyanogen bromide peptides, none of which was identical to those of protein 1, and the sum of these peptides was also equivalent to the apparent molecular weight of the uncleaved protein. Significant differences were also observed when tryptic peptides from the two proteins were compared. These results indicate that protein 1 and the phage-directed protein 2 are distinct, different, and apparently homogeneous proteins.

Amino Acids↗

PubMed1975

Physiochemical properties of DNA binding proteins: gene 32 protein of T4 and Escherichia coli unwinding protein.

The single-stranded DNA binding protein coded for by gene 32 of bacteriophage T4 and a similar protein isolated from uninfected Escherichia coli both induce characteristic changes in the circular dichroism (CD) of single-stranded nucleic acids. These CD changes have been adapted as an assay of protein-DNA complex formation. Far-ultraviolet CD spectra show the secondary structure of the two proteins to be similar with approximately 20% alpha helix, approximately 20% beta structure, and 60% random coil. Both proteins show prominent Cotton effects arising from their aromatic chromophores. Nitration of five of the nine tyrosyl residues of gene 32 protein prevents DNA binding, while prior formation of the DNA complex protects all tyrosyl residues from nitration. The tyrosyl residues may participate in gene 32 protein-DNA binding by intercalation between bases of the single strand. In contrast, no tyrosyl residues can be nitrated in the E. coli protein suggesting that surface tyrosyls do not play a part in binding of E. coli protein to DNA. Approximately 50 amino acids can be cleaved from the gene 32 protein with trypsin. This cleavage also occurs spontaneously in infected cell extracts. The remaining protein of mol wt 30000 has the same CD spectra and DNA binding properties as the native protein. The physicochemical properties can be correlated with previous work on the structures and functions of the group of DNA "unwinding proteins".

Amino Acids↗

PubMed1976

Protein synthesis with special reference to S-100 protein in brain slices from rats receiving a restricted protein supply.

The synthesis of S-100 protein and that of soluble and total proteins was investigated using cerebral slices from rats fed a 20% or 3% protein containing diet for 6 days. Incorporation of radioactive amino acids into S-100 protein was significantly higher when rats were fed a diet containing 20% protein. No significant differences were obtained in the radioactivity incorporated into total or soluble proteins between the 2 dietary groups. 14C-leucine of aspecific radioactivity of 55 mCi/mmol or 3.2 mCi/mmol incorporated with time into total protein was similar for the 2 dietary groups. The time-dependent uptake of 14C-leucine by the slices and theinulin space remained unaffected by the dietary conditions used; and amino acid analyser estimates of the free amino acid pool showed no significant differences. Brain wet weight was 1.54+/-0.02 g and1.39+/-0.02 g for protein-fed and protein-restricted rats respectively. The corresponding body weight increased by 7.8 g/day or fell by 0.5 g/day. Although the differences observed in total protein synthesis were small the synthesis of a nervous tissue specific protein S-100 was markedly affected by short-term protein restriction.

Amino Acids↗

PubMed1975

Protein metabolism in hypo- and hyperstimulated rat thyroid glands. I. Protein synthesis of different thyroidal proteins.

The comparative study of the in vivo synthesis of thyroglobulin and proteins other than thyroglobulin was carried out in thyroid glands from animals submitted to different levels of TSH stimulation. The different levels of hormonal stimulation modify neither the rate of labeling after injection of the isotope, nor the level of the free labeled amino acid in the glands (percent of the total uptake), but they have a very significant effect on the level of incorporation of the isotope into total proteins. In hypostimulated thyroids the total protein synthesis is very much reduced, while in hyperstimulated glands it is significantly increased. In both hyper- and hypostimulated animals, the proportion of radioactivity bound to the particulate protein fraction is higher than in control rats. However, the solubilization by digitonine of these proteins is lower in hypostimulated and higher in hyperstimulated animals than in controls. Thyroglobulin synthesis is significantly modified qualitatively and quantitatively in both hypo- and hyperstimulated glands. Qualitative modifications are characterized by a changed ratio of 19 S/12 S molecules with respect to the controls. This is probably caused by a more important dissociation of 19S molecules, due to the lower level of halogenation in both hypo- and MTU treated glands. The quantitative modifications of thyroglobulin synthesis, expressed either in absolute values (DPM/mg of tissue), or relatively to the total proteins (percent of total newly formed proteins), are characterized by a very important inhibition of this synthesis in hypostimulated glands, and its stimulation in glands chronically submitted to the TSH action. The modifications of synthesis observed for the proteins other than thyroglobulin are less significant in both types of treated glands than are those observed for thyroglobulin. The level of hormonal stimulation has no effect on the distribution of these proteins between soluble and the particulate fraction, but seems to have a slight effect on the solubilization of the latter ones. Comparative evaluation of the TSH effect on the synthesis of different thyroidal proteins shows that it has a much more specific and significant action on thyroglobulin than on other proteins. The differential effect of TSH on the synthesis of thyroglobulin and proteins other than thyroglobulin suggests that different mechanisms may exist by which TSH regulates the synthesis of these two types of proteins.

Animals↗

PubMed1979

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.

Alleles↗

PubMed1977

Regulation of Protein Synthesis in Hen's oviducts. I. Removal of extracellular and intracellular proteins and their effects on protein synthesis.

The minced oviducts prepared from laying hens contained considerable amounts of extracellular proteins (about 60 mg per g of the wet oviducts), which could largely be removed (about 80%) by washing four times with chilled 5rebs-Ringer-Tris buffer, pH 7.6. When the washed, minced oviducts were incubated at 37 degrees with the buffer, intracellular proteins were increasingly secreted to the extracellular fluids as the incubation time was increased and the amounts of intracellular proteins decreased concomitatnly, suggesting that the rate of secretion of intracellular proteins was faster than that of protein synthesis under the conditions used (without addition of amino acids to the medium). Repeated 5-min incubations (five to seven times) resulted in the secretion of intracellular proteins in amounts of 100 mg or more per g of the wet oviducts. The secretion rate of intracellular proteins was almost constant at 77 degrees regardless of the volume of buffer used for the incubation, and no secretion occurred at 4 degrees even after 5 h. It appeared that a 30 min period was required for the secretion of intracellular proteins from the oviducts at 37 degrees and that the rate of secretion was not affected appreciably by the amount of secreted proteins present in the extracellular fluids. The protein synthesis activity in the washed, preincubated minced oviducts was independent of the amount of intracellular proteins involved but was dependent upon that of secreted proteins present in the extracellular fluids.

Animals↗

PubMed1978

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).

Chemical Phenomena↗