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:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recent introduction of a reliable, T-lymphocyte proliferation assay, which utilizes thioglycollate-induced, nylon wool column-passed, peritoneal exudate lymphocytes from immune mice (PETLES), allowed us to investigate the genetic control of murine immune responses at the T-lymphocyte level. Examination of the blast cells generated in this population 5 days after stimulation with antigen, revealed that 85% of the cells bore the Thy 1 antigen on their surface, whereas only 5% bore immunoglobulin. Thus, the assay can be considered to measure almost exclusively T-lymphocyte function. This assay was used to examine the T-lymphocyte proliferative responses to seven different antigens: poly(Glu60Ala30Tyr10), poly(Glu58Lys38Tyr4), poly-(Tyr,Glu)-poly-D,L-Ala--poly-Lys, poly-(Phe,Glu)-poly-D,L-Ala--poly-Lys, staphylococcal nuclease, lactate dehydrogenase H4, and the BALB/c IgA myeloma protein, TEPC-15. PETLES from a large number of different inbred mouse strains, including H-2 congenic resistant lines and H-2 recombinants, were studied. The strains could be classified as high responders, low responders, or nonresponders to a particular antigen as judged by the magnitude of the T-lymphocyte proliferative response. In every case but one this classification corresponded to the responder status given the strain based on its ability to mount an in vivo antibody response to the same antigen. For two of the antigens, poly-(Tyr,Glu)-poly-D,L-Ala--poly-Lys and TEPC-15, the immune response genes controlling the T-lymphocyte proliferative response were mapped to the K region or I-A subregion of the major histocompatibility complex, as had previously been shown for the control of the antibody responses to these antigens. This tight linkage of the two phenotypic responses very strongly suggests that the same immune response gene controls the expression of both the proliferative and antibody responses. Since there is essentially no contribution from B lymphocytes in the T-lymphocyte proliferation assay, it seems reasonable to conclude that none of the seven immune response genes studied are expressed solely in B lymphocytes.
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PMID:T-lymphocyte-enriched murine peritoneal exudate cells. II. Genetic control of antigen-induced T-lymphocyte proliferation. 108 91

The recent development of a reliable murine T lymphocyte proliferation assay has facilitated the study of T lymphocyte function in vitro. In this paper, the effect of anti-histocompatibility antisera on the proliferative response was investigated. The continuous presence of anti-Ia antisera in the cultures was found to inhibit the responses to the antigens poly (Glu58 Lys38 Tyr4) [GLT], poly (Tyr, Glu) ploy D,L Ala-poly Lys [(T,G)-A--L], poly (Phe, Glu)-poly D,L Ala-poly Lys [(phi, G)-A--L], lactate dehydrogenase H4, staphylococcal nuclease, and the IgA myeloma protein, TEPC 15. The T lymphocyte proliferative responses to all of these antigens have previously been shown to be under the genetic control of major histocompatibility-linked immune response genes. The anti-Ia antisera were also capable of inhibiting proliferative responses to antigens such as PPD, to which all strains respond. In contrast, antisera directed solely against H-2K or H-2D antigens did not give significant inhibition. Anti-Ia antisera capable of reacting with antigens coded for by genetically defined subregions of the I locus were capable of completely inhibiting the proliferative response. In the two cases studied, GLT and (T,G)-A--L, an Ir gene controlling the T lymphocyte proliferative response to the antigen had been previously mapped to the same subregion as that which coded for the Ia antigens recognized by the blocking antisera. Finally, in F1 hybrids between responder and nonresponder strains, the anti-Ia antisera showed haplotype-specific inhibition. That is, anti-Ia antisera directed against the responder haplotype could completely block the antigen response controlled by Ir genes of that haplotype; anti-Ia antisera directed against Ia antigens of the nonresponder haplotype gave only partial or no inhibition. Since this selective inhibition was reciprocal depending on which antigen was used, it suggested that the mechanism of anti-Ia antisera inhibition was not cell killing or a nonspecific turning off of the cell but rather a blockade of antigen stimulation at the cell surface. Furthermore, the selective inhibition demonstrates a phenotypic linkage between Ir gene products and Ia antigens at the cell surface. These results, coupled with the known genetic linkage of Ir genes and the genes coding for Ia antigens, suggest that Ia antigens are determinants on Ir gene products.
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PMID:T lymphocyte-enriched murine peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation with anti-Ia antisera. 108 2

Results from a number of recent studies suggest that amino acid insertion mutations may provide an important alternative to substitution mutations for modifying protein structures and functional activities. To facilitate the use of single-amino acid insertions, we have developed a general strategy for inducing random, in-phase codon insertions across a defined segment of a cloned gene. In brief, a mixture of blocked and protected trinucleotide phosphoramidites is coupled at substoichiometric levels after every third monomer coupling on a conventional solid-state synthesizer. From the heterogeneous mixture of oligonucleotide sequences thus generated, those oligonucleotides that have acquired a single additional codon are purified by urea/PAGE. By using equimolar amounts of GCT and GGT trinucleotides in the oligonucleotide synthesis plus standard oligonucleotide-directed mutagenesis techniques, we have induced as many as 13 different single alanine and glycine insertion mutations into the gene for staphylococcal nuclease in one experiment. On replacement of the 5'-dimethoxytrityl blocking group on the trinucleotide phosphoramidite with an acid-stable blocking group, such as levulinate or fluoren-9-ylmethoxycarbonyl (Fmoc), this same strategy of substoichiometric couplings at codon boundaries should permit the synthesis of complex pools of oligonucleotides for the introduction, with constant efficiency, of every type of amino acid substitution at each codon across a gene segment.
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PMID:A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. 156 54

In order to quantitate the contributions of the polar, uncharged amino acids to the stability of the native state of staphylococcal nuclease, each of the 13 alanines, 9 glycines, 9 threonines, 6 prolines, 6 glutamines, 6 asparagines, and 3 serines was substituted, either with both alanine and glycine or with 1 of these 2 amino acids plus valine. For each mutant, the stability to reversible denaturation (delta GH2O) was quantitated by determining the Kapp for this reaction as a function of guanidine hydrochloride concentration. In addition, the parameter mGuHCl (= d(delta G)/d[GuHCl]) was calculated from the data. To identify the local structural features responsible for the relatively large and variable changes in delta GH2O and mGuHCl observed for the same type of substitution at different locations in nuclease, statistical correlations were sought between delta GH2O, mGuHCl, and a number of descriptors of the local structure. As with substitutions of the large hydrophobic amino acids [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041], mutation of polar, uncharged residues to Gly leads to a change in stability that, on average, correlates well with the degree to which the wild-type residue is buried. This correlation is especially significant for threonine, an amino acid with both polar and hydrophobic character, but is not demonstrated for the more typically hydrophobic residue alanine. As reported in the previous study of alanine/glycine substitutions of hydrophobic residues, a significant correlation between changes in stability and changes in the value of mGuHCl is again observed, strengthening the conclusion that the putative structural changes in the denatured state which lead to increases or decreases in mGuHCl are responsible for a significant fraction of the stability loss for an average mutant. The existence of this correlation is consistent with the denatured state of wild-type staphylococcal nuclease having evolved to a relatively high free energy via optimization of a balance between a maximal exposure of hydrophobic surface and a minimal gain in chain entropy. On average, mutations are less stable in proportion to the extent of which they perturb this balance. A new and puzzling correlation is reported between the extent of buriedness of a residue in the wild-type native state versus the difference in mGuHCl between the Ala mutation and the Gly mutation at that position.
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PMID:Contributions of the polar, uncharged amino acids to the stability of staphylococcal nuclease: evidence for mutational effects on the free energy of the denatured state. 161 Aug 20

In a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:299-305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl-glycine, glycyl-glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non-additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl-glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural and energetic differences between insertions and substitutions in staphylococcal nuclease. 162 Jun 95

On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.
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PMID:In vivo effect of asparagine in the hydrophobic region of the signal sequence. 186 Aug 48

To quantitate the contributions of the large hydrophobic residues in staphylococcal nuclease to the stability of its native state, single alanine and glycine substitutions were constructed by site-directed mutagenesis for each of the 11 leucine, 9 valine, 7 tyrosine, 5 isoleucine, 4 methionine, and 3 phenylalanine residues. In addition, each isoleucine was also mutated to valine. The resulting collection of 83 mutant nucleases was submitted to guanidine hydrochloride denaturation using intrinsic tryptophan fluorescence to monitor the equilibrium constant between the native and denatured states. From analysis of these data, each mutant protein's stability to reversible denaturation (delta GH2O) and sensitivity to guanidine hydrochloride (mGuHCl or d(delta G)/d[GuHCl]) were obtained. Four unexpected trends were observed. (1) A striking bipartite distribution was found for sites of mutations that altered mGuHCl: mutations that increased this parameter only involved residues that contribute side chains to the major hydrophobic core centered around a five-strand beta-barrel, whereas mutations that caused mGuHCl to decrease clustered around a second, smaller and less well-defined hydrophobic core. (2) The average stability loss for mutants in each of the six residue classes was 2-3 times greater than that estimated on the basis of the free energy of transfer of the hydrophobic side chain from water to n-octanol. (3) The magnitude of the stability loss on substituting Ala or Gly for a particular type of amino acid varied extensively among the different sites of its occurrence in nuclease, indicating that the environment surrounding a specific residue determines how large a stability contribution its side chain will make.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contributions of the large hydrophobic amino acids to the stability of staphylococcal nuclease. 226 61

Single alanine and glycine insertions were introduced at 20 randomly selected positions in staphylococcal nuclease. The resulting changes in catalytic activity and in stability to guanidine hydrochloride denaturation indicate that the native state structure is frequently able to accommodate the extra residue without great difficulty, even insertions within secondary structural elements such as alpha helices and beta sheets. On average, an inserted residue reduces the free energy of denaturation (delta GH2O) by an amount roughly comparable to an alanine or glycine substitution for one of the residues flanking the site of insertion. Several positions outside of the enzyme active site were found where insertions, but not substitutions, lead to structural changes that modify catalytic activity and the circular dichroism spectrum. Amino acid insertions represent a virtually unexplored class of genetic mutation that may prove complementary to amino acid substitutions for engineering proteins with altered functional and structural properties.
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PMID:Accommodation of single amino acid insertions by the native state of staphylococcal nuclease. 238 4

The high-resolution X-ray crystal structure of staphylococcal nuclease suggests that the gamma-carboxylate group of Glu-43 is directly involved in catalysis as a general base that facilitates the attack of water on the substrate phosphodiester. We have used primer-directed, site-specific mutagenesis to generate aspartate, glutamine, asparagine, alanine, and serine substitutions for this residue. The Vmax/Km for the aspartate mutant is reduced 1400-fold and the values for the charge-neutral mutations are reduced 5000-fold relative to the wild-type enzyme. Although these reductions in catalytic efficiency might appear useful in quantitatively estimating the importance of general basic catalysis in the reaction catalyzed by the wild-type enzyme, the thermal stabilities and 1H NMR spectral properties of the mutants suggest that such interpretations are ambiguous. All five mutants have higher melting temperatures for thermal denaturation than the wild-type enzyme, suggesting that the mutants have enhanced thermal stabilities relative to the wild-type enzyme. Chemical shift changes relative to the wild type are observed in both the aromatic and upfield-shifted methyl group regions of the 1H NMR spectra of the aspartate and serine mutants, suggesting the presence of conformational differences between the wild-type and mutant enzymes. That these conformational differences may be large enough to be mechanistically relevant is suggested by comparisons of the magnitudes of nuclear Overhauser effect (NOE) correlations between the aromatic and upfield-shifted methyl group regions observed via two-dimensional nuclear Overhauser effect correlation spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutants of staphylococcal nuclease. Detection and localization by 1H NMR spectroscopy of conformational changes accompanying substitutions for glutamic acid-43. 289 75

Oligonucleotide-directed site-specific mutagenesis was used to systematically shorten the hydrophobic region within the signal peptide of the Escherichia coli outer membrane protein OmpA. DNA encoding the wild type and mutant OmpA signal peptides were then fused in frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. The ability of these signal peptides to direct processing of the resulting hybrid proteins was dependent on both their length and the protein to which they were fused. Deletion of two or more residues progressively slowed processing of pro-OmpA-nuclease. By contrast, pro-OmpA-beta-lactamase was less sensitive to the length of the hydrophobic region than to the nature of the deleted residue(s). Deletion of an Ala residue tended to reduce processing efficiency of pro-OmpA-beta-lactamase, while deletion of an Ile residue, together with the Ala residue, resulted in improvement. The loss of either 3 or 4 residues abolished processing of both hybrids. These data indicate that both the length as well as the identity of residues in the hydrophobic region are important. The relative importance of these two factors depends on the mature region of the protein being secreted.
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PMID:The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide. 354 10


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