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)

We have sought to determine how much amino acid diversity is tolerable at position 69 of the Ak alpha chain, a position previously implicated as a peptide contact site. Slot-machine mutagenesis was used to create a set of 11 mutant Ak alpha cDNAs, each specifying a different amino acid at position 69. These cDNAs were individually expressed in L cells together with a wild-type Ak beta cDNA to produce a panel of mutant antigen-presenting cell lines. The ability of each member of this panel to present a hen egg lysozyme and a bovine ribonuclease peptide to various T hybridomas was assessed. We found that a surprising degree of amino acid diversity is tolerable at Ak alpha position 69: even charged (Glu, Arg) or bulky (Trp, Tyr) residues can be accommodated without abrogating cell-surface expression of Ak, peptide binding to it, or T cell recognition of it. We discuss the implications of these findings for models of T cell recognition of the class II molecule/antigen duplex.
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PMID:Local structure of a peptide contact site on Ak alpha. 191 51

Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.
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PMID:Demonstration by NMR of folding domains in lysozyme. 200 Jan 38

The binding of immunogenic peptides to class II major histocompatibility molecules was examined at various pH values. We studied binding of peptides containing residues 52-61 from hen egg lysozyme (HEL) to I-Ak on fixed peritoneal macrophages or to solubilized affinity-purified I-Ak. Optimum binding occurred at pH 5.5-6.0 with accelerated kinetics relative to pH 7.4; equilibrium binding was also higher at pH 5.5-6.0 than at 7.4. Similar enhancement at pH 5-6 was observed for the binding of hemoglobin-(64-76) to I-Ek and of ribonuclease-(41-61) to I-Ak. In contrast, the binding of HEL-(34-45) to I-Ak was minimally enhanced at acid pH. Dissociation of cell-associated or purified peptide-I-Ak complexes was minimal between pH 5.5 and 7.4, with increased dissociation only at or below pH 4.0 [HEL-(46-61)] or pH 5.0 [HEL-(34-45)]. Thus, optimum peptide binding occurs at pH values similar to the endosomal environment, where the complexes appear to be formed during antigen processing. In addition, we examined the effect of a number of polysaccharides on the binding of peptide to I-Ak. None of these competed with the HEL peptide 125I-labeled YE52-61 for binding to I-Ak. [3H]Dextran also failed to bind purified I-Ak. Polysaccharides do not appear to bind to class II major histocompatibility complex molecules, which explains the T-cell independence of polysaccharide antigens.
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PMID:Effects of pH and polysaccharides on peptide binding to class II major histocompatibility complex molecules. 201 83

A semi-empirical method has been used to estimate the thermodynamic parameters of hydration of buried surface areas of ribonuclease S, lysozyme and myoglobin from the model of complete unfolding according to Ooi et al. ((1987) Proc. Natl. Acad. Sci. USA 84, 3086-3090). The buried surface area of proteins is considered as the difference between the accessible surface area of native protein and the completely extended polypeptide chain according to Lee and Richards ((1971) J. Mol. Biol. 55, 379-400). The contributions of nonpolar and polar protein groups to the general value of Gibbs energy, enthalpy, entropy and heat capacity of hydration have been determined. The obtained results on the thermodynamic behavior of proteins in the process of complete unfolding are in good agreement with the results of microcalorimetric studies of thermal denaturation.
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PMID:Thermodynamic properties of globular proteins and the principle of stabilization of their native structure. 222 40

Seven cationic substances--human and egg-white lysozyme, RNase, protamine, histone, poly-L-lysine and poly-L-arginine; five cationic lysosomal fractions from human polymorphonuclears (PMNs); RNA; poly-L-glutamic acid; DNA; heparin; endotoxin; mastocytotropic agent compound 48/80; and cytochalasin B were tested for the influence on chemotaxis and random migration of human PMNs using under-agarose migration and Boyden chambers with two filters and [51Cr]PMNs. The above substances were either preincubated with PMNs, added to chemoattractants, or used instead of chemoattractants. In under-agarose migration method chemotaxis was inhibited by 11-35% when egg-white lysozyme, protamine, heparin, endotoxin, or compound 48/80 was added to the cells. High concentration of cytochalasin B inhibited chemotaxis by 73%. Cationic fractions I and V and low concentration of cytochalasin B enhanced chemotaxis by 11%, 41%, and 30%, respectively. When human and egg-white lysozyme, DNA, or cytochalasin B was added to the chemoattractants, motility of PMNs was inhibited. Cationic fractions II and V from human PMNs, when used as chemoattractants, enhanced cellular motility by 143-167%. Random migration was enhanced by heparin and inhibited by cytochalasin B and by cationic fractions from human PMNs. These findings suggest that various cationic and anionic substances and cationic fractions from human PMNs have heterogeneous influence on random migration and chemotactic activity of human PMN. Analysis relating chemotaxis to phagocytosis and to intracellular bactericidal activity (ICBA) has shown several patterns. Protamine, poly-L-lysine, poly-L-arginine, and agent compound 40/80 all inhibit chemotaxis and enhance phagocytosis and ICBA; cationic fractions II and V enhanced all three functions, whereas cytochalasin B suppressed phagocytosis and ICBA and had concentration-dependent modulatory influence on chemotaxis. It implies diverse mechanisms of action and possible impact on inflammatory reactions.
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PMID:Modulation of locomotor activity of polymorphonuclear cells by cationic substances and cationic lysosomal fractions from human neutrophils. 241 86

Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2.5) and at a chill shock temperature of 5 degrees C resulted in symptoms of injury. Cells became sensitive to 40 micrograms/ml lysozyme, 50 micrograms/ml actinomycin D and 100 micrograms/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260-280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells. The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5 degrees C, then 5 ppm chlorine at 5 degrees C and the singular chill shock stress at 5 degrees C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS. The findings obtained indicated impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.
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PMID:Mechanisms of triple stress-mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate-acidified hypochlorite system at 5 degrees C. 242

The attempt is made to find new correlations between local structural characteristics of proteins and the hydrogen exchange rates of their individual main-chain amides, and to relate such correlations to possible mechanisms of hydrogen exchange. It is found that in bovine pancreatic trypsin inhibitor (BPTI) the surface area buried by a particular residue and its neighbors correlates with the exchange rate of the main-chain amide of that residue. As the area buried by a particular fragment can be associated with the stabilization of the protein structure by this fragment, the correlation suggests a role for the energetics of the local unfolding in the mechanism of hydrogen exchange. Calculations based on the assumption that the exchange mechanism involves local unfolding lead to quantitative agreement between the calculated and experimentally measured exchange rates for 80% of the amides of BPTI that are buried or hydrogen bonded to the main-chain or to internal water molecules. The same degree of correlation is found between the calculated exchange rates and partial exchange data for ribonuclease S, hen lysozyme and cytochrome c. A similarly strong correlation is found between calculated exchange rates and the exchange rates of ribonuclease A determined by neutron diffraction in the crystal. The criteria of correlation are, however, less stringent in this case because of the experimental errors, which are larger than for solution data. It is suggested that the observed correlation be used for predictions of hydrogen exchange rates in proteins.
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PMID:Correlation between calculated local stability and hydrogen exchange rates in proteins. 244 80

The expression of the positively charged human protein secretory leukocyte protease inhibitor (SLPI) in Escherichia coli causes severe cellular toxicity. After induction of SLPI synthesis in a high-level-expression strain, SGE61, the growth of the strain is arrested and total protein and RNA synthesis rates decline by 60 to 70%. The mechanism of SLPI-mediated inhibition of macromolecular synthesis was examined in cell-free transcription-translation systems. SLPI proved to be a potent inhibitor of translation in vitro. When SLPI was added to translation reactions at SLPI/mRNA ratios attained during maximal SLPI accumulation in SGE61, translation of a test mRNA was inhibited by 75%. The mechanism of translation inhibition was deduced from in vitro experiments showing that SLPI bound to mRNA and interfered with the interaction of RNA-metabolizing enzymes, such as RNase. In addition, SLPI bound to DNA in vitro, but transcription was not inhibited as strongly in cell-free reactions as it was in SGE61. Similar nucleic acid-binding and translation inhibition properties were displayed in vitro by another basic protein, chicken egg white lysozyme, but were not displayed by the relatively acidic protein bovine serum albumin. On the basis of these results, we concluded that SLPI binds to nucleic acids via charge interactions and inhibits translation by competing with ribosomes for binding to mRNA. Since SLPI-mRNA and SLPI-DNA binding occurred at SLPI/mRNA and SLPI/DNA ratios existing in SGE61, nucleic acid binding may contribute to the toxicity of SLPI to E. coli. These results indicate that, in general, high-level expression of basic recombinant proteins in E. coli may be problematic.
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PMID:Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli. 246

There is evidence that increased excretion of urinary enzymes and low-molecular mass proteins indicate impaired tubular function. The excretion of N-acetyl-beta-D-glucosaminidase (NAG), lysozyme, and ribonuclease in Type I diabetic patients with (n = 19) and without (n = 17) persistent proteinuria (urinary protein excretion greater than 0.5 g/day) was investigated and compared with this excretion in 30 weight- and gender-matched nondiabetic subjects without renal disease. Urinary NAG excretion was significantly higher in diabetic patients with and without persistent proteinuria (1.16 +/- 0.09 and 3.19 +/- 1.2 Umol/L creatinine, respectively) compared to controls (0.37 +/- 0.03 Umol/L creatinine p less than 0.01). In addition, the urinary excretion of lysozyme and ribonuclease was significantly increased in diabetic patients. Urinary NAG was found to correlate positively with albuminuria and proteinuria (r = 0.95 and 0.93, respectively), as well as with ribonuclease and lysozyme (r = 0.93 and 0.60; p less than 0.01) in patients with persistent proteinuria. Furthermore, NAG excretion was significantly related to the duration of diabetes (r = 0.36; p less than 0.05). No relationship existed between urinary NAG and serum creatinine, beta-2-microglobulin, and degree of metabolic control (HbA7). The lysozyme excretion, but not NAG excretion, was significantly related to hypertension in patients with clinical proteinuria. In conclusion, our results suggest a relationship between the development of tubular dysfunction and the impairment of glomerular function in diabetic nephropathy. An increased excretion of NAG and low-molecular mass proteins may indicate early nephropathy
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PMID:Further evidence for tubular dysfunction in insulin dependent diabetes. 252 61

A simple and cheap method of plasmid DNA preparation from both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) organism is presented here. In this method, in place of the high-priced chemicals lysostaphin and lysozyme which are commonly used for removal of cell-wall during plasmid DNA preparation from gram-positive and gram-negative bacteria, respectively, only sucrose has been used. Firstly, bacteria is treated with Trizma (pH 8.0) containing 100% sucrose (hypertonic solution). Due to this osmotic shock, protoplasm covered by the plasma membrane of bacteria possibly shrinks and becomes detached from the cell-wall. Osmotically sensitive cells thus formed, from gram-positive (S. aureus) and gram-negative (E. coli) bacteria, are finally lysed by the lysis mixture, containing brij 58 and sodium deoxycholate. The lysate is centrifuged at 15,000 rpm for 30 min to pellet the cell debris. The supernatant containing plasmid DNA is treated with either polyethylene glycol or isopropanol. The precipitate which contains plasmid DNA is dissolved in a buffer containing Tris, EDTA, NaCl, and sodium dodecyl sulfate (pH 8.0); thus protein is denatured and removed. Finally, RNA is removed by RNase treatment. The average yield of staphylococcal plasmid DNA as well as plasmid pBR322 from E. coli HB101 in 100% sucrose-treated preparations is greater than that of lysostaphin- and lysozyme-treated preparations. This method is applicable for both large-scale and small-scale preparations. The substrate activity for restriction enzyme, cloning, transforming ability, and electron microscopic profile of the plasmid DNA prepared by this method remains unaltered.
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PMID:A new method of plasmid DNA preparation by sucrose-mediated detergent lysis from Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive). 254 9


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