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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.
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PMID:Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system. 300 81

A soluble ATP/Mg2-dependent proteolytic system from rabbit cardiac muscle has been identified (m ca. 310 kDa) and purified ca. 9-fold. This enzyme which splits the substrate [3H]globin and 125I-bovine serum albumin (125I-BSA) has many similarities to the ATP-dependent proteolytic enzyme system from reticulocytes which utilizes ubiquitin: 1) The specific activities in reticulocyte lysates and cardiac muscle extracts are of the same magnitude (0.5-1 arb. unit/mg). 2) The binding and elution behavior on DEAE-cellulose is similar. 3) In both cases the pH optimum (substrate 125I-BSA) is pH 7.6. 4) Both enzymes are inhibited by hemin, NEM and iodoacetate but not e.g. by leupeptin, or inhibitors of serine proteases. 5) Neither enzyme system can utilize ATP-analogs such as AMP-CPP, AMP-PCP, AMP-PNP or ATP-gamma-S. There are however also significant differences: 1) The enzyme system from cardiac muscle is fully active in the absence of ubiquitin and cannot be activated by this peptide. 2) The enzyme from cardiac muscle can degrade methylated BSA. 3) The cardiac muscle enzyme can be further purified on Sepharose 4B; the enzyme from reticulocytes is inactivated by this procedure. 4) The cardiac enzyme cannot be inactivated by ribonuclease as the reticulocyte counterpart. Although ubiquitin does not appear to play a role in the isolated ATP/Mg2-dependent proteolytic system from cardiac muscle, it is demonstrated for the first time that 125I-ubiquitin can be conjugated to a wide variety of cardiac muscle proteins in vitro in an ATP-dependent manner. Apparent molecular masses of major conjugates were: 185 kDa, 140 kDa, 85 kDa, 65 kDa, 46 kDa, 38 kDa and 36 kDa as estimated by discontinuous SDS gel electrophoresis. Addition of purified phosphorylase kinase to cardiac muscle extract changed the ubiquitination pattern by the appearance of two novel protein bands. It is concluded that the ATP/Mg2-dependent proteolytic system of cardiac muscle must be differentiated from the proteolytic system of reticulocytes mainly because of its ubiquitin-independence. Nevertheless the conjugation of 125I-ubiquitin to many muscle proteins is a strong indication for a crucial role of this interesting peptide in striated muscle.
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PMID:ATP-dependent proteolysis and the role of ubiquitin in rabbit cardiac muscle. 304 36

The effects of both 3,5,3'-triiodo-L-thyronine and spontaneous metamorphosis on Rana catesbeiana liver mRNA were studied using in vitro translation of isolated liver poly(A)+ RNA in a rabbit reticulocyte lysate system. Conventional phenol extraction methods yielded degraded RNA due to high levels of endogenous ribonucleases released upon homogenization of Rana catesbeiana liver. Isolation of intact total RNA was achieved using the potent ribonuclease denaturant, guanidinium thiocyanate. Adult bullfrog serum albumin was purified to homogeneity and a monospecific antibody was elicited against it. A serum protein of 23,000 daltons that migrated near serum albumin on a 6% native gel was also purified to homogeneity. A monospecific antibody was also raised against this protein. Both antibodies were used to quantitatively immunoprecipitate the in vitro translation products of poly(A)+ RNA isolated at intervals following a single injection of triiodothyronine or during various stages of spontaneous amphibian metamorphosis. Triiodothyronine caused a sevenfold increase in translatable albumin mRNA and a threefold increase in translatable mRNA for the 23,000 dalton protein. These increases are consistent with a nuclear initiated mechanism for thyroid hormone action during amphibian metamorphosis.
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PMID:Triiodothyronine increases translatable albumin messenger RNA in Rana catesbeiana tadpole liver. 314 56

A single stained band containing approximately 5 micrograms of protein was cut out of a polyacrylamide gel and subjected to hydrolysis together with the gel. The hydrolysate was subsequently analyzed for its amino acid content by high-performance liquid chromatography and postlabeling with o-phthalaldehyde. Bovine serum albumin, ribonuclease B, ovalbumin, pepsin, and chymotrypsinogen A were analyzed by this method, and their amino acid compositions were found to be in good agreement with the reported values. By this method, it is possible to quantitate 16 amino acids: Asx, Thr, Ser, Glx, Pro, Cys, Gly, Ala, Val, Ile, Leu, Tyr, Phe, His, Lys, and Arg. Thioglycolic acid is effective protection against the decomposition of Tyr, Cys, and Met; however, the recovery of Met is inconsistent. This method might be very helpful for the amino acid analysis of proteins of multicomponent systems, especially, those which can be resolved only by polyacrylamide gel electrophoresis.
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PMID:Amino acid analysis by high-performance liquid chromatography of a single stained protein band from a polyacrylamide gel. 357 64

Fourier transform-infrared (FT-IR) spectra are reported for the amide III spectral region of the native and thermally denatured forms of chymotrypsinogen, ribonuclease, bovine serum albumin, and lysozyme. Chymotrypsinogen denatures into structures containing substantial contributions from beta-sheets, while lysozyme and bovine serum albumin show increased amounts of random-coil forms. The changes observed for ribonuclease are quite small. Bovine serum albumin shows at least six bands in the 1,260-1,320 cm-1 region which undergo large intensity changes upon thermal denaturation, and hence are assignable to alpha-helical amide III modes. The large number of observed bands suggests that slight variations in helical geometry, symmetry, or interactions result in changed amide III frequencies, so that simple correlations between narrow frequency ranges and secondary structures may not be applicable for this mode. A widened frequency range is suggested as diagnostic for helical structures.
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PMID:Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amide III spectral region. 360 22

The neurotoxic gamma-diketone, 2,5-hexanedione, reacts with axonal protein amine residues to form 2,5-dimethylpyrrole adducts. Current evidence implicates this reaction as the potentially critical step in gamma-diketone neurotoxicity, although it is unclear whether pyrrole formation per se is sufficient to induce neuropathy or whether secondary autoxidative reactions are also required. The present in vitro study examines aspects of pyrrole formation and the secondary phenomena of chromophore development and covalent protein crosslinking in 2,5-hexanedione-treated protein. p-Dimethylaminobenzaldehyde (DMAB)-detectable pyrrole concentrations decreased linearly with time when pyrrolylated bovine serum albumin (pyrrole-BSA) was incubated under air, but remained unchanged following N2 incubation. The air-induced decrease was accompanied by the appearance of chromophores and crosslinked protein. Covalent crosslinking of pyrrole-BSA was pH-dependent, with relatively increased intermolecular bridging at pH 7.4 as compared to pH 9.5. Chromophore formation and the loss in DMAB-detectable pyrrole were also accelerated at the lower pH. Autoxidative parameters were inhibited in the presence of a free radical scavenger (ascorbic acid) but induced by free radical initiators (potassium persulfate and 2,2'-azobis[2-amidinopropane hydrochloride]). In vitro incubation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of combinations of bovine serum albumin, ribonuclease, pyrrole-BSA, and pyrrolylated ribonuclease revealed that the intermolecular crosslinking pathway was mediated by pyrrole-pyrrole bridging. These findings demonstrate that the secondary autoxidative phenomena following pyrrole adduct formation in gamma-diketone-treated protein proceed via pH-dependent, free radical-mediated mechanisms. If similar mechanisms are present in vivo, the results also suggest that intermolecular covalent crosslinking of pyrrolylated axonal protein may be less widespread and more specific than previously thought.
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PMID:Mechanisms of in vitro pyrrole adduct autoxidation in 2,5-hexanedione-treated protein. 377 83

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.
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PMID:Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein. 397 26

Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.
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PMID:Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium. 401 90

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.
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PMID:Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts. 404 85

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.
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PMID:Effect of some proteins on the yeast cell membrane. 429 20


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