Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The circulating half-lives of the four isozymes of bovine pancreatic ribonuclease (RNases A, B, C, and D) have been determined in normal and in nephrectomized rats. The isozymes differ only in their glycosyl content. While A contains no sugars, B has a simple oligosaccharide (GlcNAc2 Man4-5),and C and D each have a complex oligosaccharide (GlcNAc4 Man 2-3 Gal2 Fuc NeuAc2, and GlcNAc4 Man3 Gal2 Fuc NeuAc4, respectively) attached to Asn-34 of the polypeptide chain. All four isozymes were cleared rapidly in normal rats (t 1/2 = 2 to 3 min), as expected on the basis of the established role of the kidneys in removing low molecular weight proteins from circulation. In nephrectomized rats, however, a much slower clearance was observed, thus permitting the evaluation of the role of the carbohydrate chains in the catabolism of the isozymes. The clearance curves can be analyzed in terms of two processes, a rapid initial one, shown to represent the equilibration of the injected enzyme into extravascular space, and a second one which is interpreted as the catabolic clearance of the enzyme. The haf-life of the RNase isozymes was calculated from this second process and found to be in the range 528 to 577 min for RNase A, 15 min for RNase B, 681 to 862 min for RNase C, and 839 to 941 min for RNase D. The rapidly cleared RNase B was treated with alpha-mannosidase to remove 3 of the 4 mannosyl residues, leaving only a trisaccharide (GlcNAc2-betaMan) attached to the protein. The half-life of this RNase B derivatives was found to be in the range 616 to 733 min. From these results it is concluded (a) that the addition of complex oligosaccharides to a protein does not have any significant direct effect on its circulating half-life (RNases C and D compared to RNase A), and (b) that in the rat there exists a mechanism for clearing glycoproteins based on specific recognition of exposed alpha-mannosyl residues (RNase B compared to the other isozymes and to alpha-mannosidase-treated RNase B).
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PMID:Effect of glycosylation on the in vivo circulating half-life of ribonuclease. 97 51

Membrane fraction RNA isolated from rat pituitary tumor (GC) cells has been translated in a wheat germ extract. A product was synthesized which was immunologically related to growth hormone, but which migrated more slowly than growth hormone upon sodium dodecyl sulfate-acrylamide gel electrophoresis. The mobility of the cell-free product on gels of this type was unchanged by treatment with either KOH or RNase. The mobilities during paper electrophoresis of the methionine-containing tryptic peptides obtained from the cell-free product were identical to those obtained from growth hormone synthesized and secreted by the GC cells. Molecular weights for growth hormone and the cell-free product of 19,500 and 24,000, respectively, were determined by gel electrophoresis of these proteins together with marker proteins of known molecular weights. No protein with the properties of the cell-free product was detected after a 2 min incubation of the GC cells with [35S]methionine. However, treatment of the GC cells, with a protease inhibitor, L-1-tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK), led to the appearance of a new polypeptide, immunologically related to growth hormone, and with a mobility on gels identical to that of the cell-free product. These results strongly imply that the cell-free product represents a growth hormone precursor (pregrowth hormone) which is rapidly converted to growth hormone in pituitary cells.
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PMID:Pregrowth hormone: product of the translation in vitro of messenger RNA coding for growth hormone. 106 Nov 24

Cells of Escherichia coli selectively degrade proteins that have incorporated amino acid analogs. Within 1 hour after exposure of cells to canavanine, 50% of the analog-containing proteins were degraded to acid-soluble form. At the same time, no net loss of canavanine-containing protein occurred from the 100,000 X g supernatant. Instead, most of the proteins containing the analog, unlike normal ones, accumulated in particulate fractions sedimenting at 10,000 X g or 100,000 X g. They were then lost from these fractions concomitant with the degradation of the abnormal proteins. The loss of such proteins from particulate fractions accounted for all of the protein degraded to acid-soluble form. Similar observations were obtained after incorporation of other analogs or puromycin. The 10,000 X g pellets correspond to amorphous dense intracellular granules visible in electron micrographs of cells exposed to canavanine. Upon removal of the analog, these granules disappeared, simultaneously with the degradation of the analog-containing proteins. These pellets do not resemble a degradative organelle, like the lysosome; they are not osmotically sensitive, do not exclude inulin, are not enclosed by a membrane, and do not show autolytic activity. The proteins in the granules could be solubilized by sodium dodecyl sulfate but not by Triton, NaC1, dithiothreitol, RNase, DNase, or phospholipase. The proteins extracted from the pellet with sodium dodecyl sulfate tend to become particulate again upon removal of this detergent. Incorporation of canavanine caused a normally soluble polypeptide, the monomer of beta-galactosidase, to be inactive and found in the sedimentable fraction. These findings suggest that (a) the presence of amino acid analogs in proteins can make them less soluble, and (b) the inclusions are formed by the spontaneous precipitation of abnormal proteins rather than by an active granule-forming process.
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PMID:Degradation of abnormal proteins in Escherichia coli. Formation of protein inclusions in cells exposed to amino acid analogs. 108 51

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.
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PMID:Regulation of mitochondrial protein synthesis at the polyribosomal level. 110 23

A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.
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PMID:A synthetic 70-amino acid residue analog of ribonuclease S-protein with enzymic activity. 111 95

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The state of mRNA and monomeric ribosomes after the polysome dissociation was studied. The mRNA was selectively labeled with [14C] orotate after a low dose of actinomycin D. Sucrose density gradient centrifugation of Triton X-100-treated cytoplasm revealed an accumulation of heterodisperse radioactive material with very large S values. This material was converted to smaller S values with deoxycholate treatment and was extremely sensitive to mild ribonuclease treatment. Since this material was banded at around 1.43 g/cm3 in CsCl gradient centrifugation and contained RNA with a distribution of S values characteristic of polysomal mRNA, this material was identified as mRNA-containing ribonucleoprotein particles. The monomeric ribosomes were shown to be dissociated into subunits in the presence of 0.5 M KCl, indicating that these lacked nascent polypeptide chains. When the animals were recovered from the ethionine treatment by subsequent administration of adenine and methionine, the heterodisperse ribonucleoprotein particles and monomeric ribosomes appeared to be utilized for the reformation of polysomes.
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PMID:The state of messenger ribonucleic acid and ribosomes in the cytoplasm of ethionine-treated rat liver. 111 2

Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.
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PMID:Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts. 113 14

To clarify the mode of interaction between sodium dodecyl sulfate (SDS) and protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis, the binding of SDS to several protein polypeptides was investigated by the equilibrium dialysis technique. Each of the binding isotherms was characterized by the presence of two phases: an initial gradual increase in the amount of binding to 0.3-0.6 g/g (first phase) and a subsequent steep increase to 1.2-1.5 g/g (second phase). The binding was completed at a concentration of SDS below the critical micelle concentration. Throughout the first and second phases, the isotherms obtained were different for each kind of protein. On the basis of experiments with bovine serum albumin and ribonuclease (EC 3.1.4.22], the isotherms were profoundly affected by the method used for modification of the sulfhydryl groups. The claim of Reynolds and Tanford (Proc. Natl, Acad. Sci. U.S., 66, 1002 (1970)) that the isotherms are virtually identical for many kinds of proteins was not supported by the present data. Changes in the gross and local conformations were examined with reference to the isotherms by measurements of CD spectrum, free boundary electrophoresis, and gel filtration. The results obtained were collectively interpreted based on the model of SDS-protein polypeptide complexes proposed by the present authors (J. Biochem., 75, 309 (1974)).
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PMID:Binding isotherms of sodium dodecyl sulfate to protein polypeptides with special reference to SDS-polyacylamide gel electrophoresis. 115 59

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.
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PMID:Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii. 116 19

Dromedary (Camelus dromedarius) RNAase (ribonuclease) was isolated from pancreatic tissue by affinity chromatography. Peptides obtained by digestion with different proteolytic enzymes and CNBr were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. Peptides were sequenced by the dansyl-Edman method. All peptide bonds were overlapped by one or more peptides. The polypeptide chain consists of 123 amino acids. A deletion (position 39) was observed in an external loop of the polypeptide chain (residues 35-40), as was found earlier to horse RNAase (Scheffer & Beintema, 1974). A heterogeneity was found at position 103 (glutamine and lysine). Dromedary RNAase differs at 23-32% of the positions from all other pancreatic RNAases sequenced to date. In evolutionary terms this indicates that dromedary RNAase has evolved independently during the larger part of the evolution of the mammals. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50046 (14 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.
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PMID:The amino acid sequence of dromedary pancreatic ribonuclease. 116 57


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