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At least 73 records · Page 4Linked to original sources
PubMed1996

Inhibition of Serratia marcescens nuclease secretion by a truncated nuclease peptide.

The production of extracellular nuclease (Nuc) from the Serratia marcescens nucA chromosomal locus is inhibited in cells producing the N-terminal portion of Nuc from a multicopy plasmid. This inhibition in trans is not at the level of nucA expression, but rather at the level of secretion of the Nuc protein. Production of the periplasmic protein beta-lactamase (Bla) does not inhibit Nuc production unless fused to the nucA signal peptide and expressed from nucAp. Inhibition by either the truncated Nuc peptide (delta Nuc) or a Bla fusion protein is promoter specific and observed when expressed from nucAp; little inhibition is observed when the same protein is expressed from the lacZpo promoter-operator. This promoter specificity is also true for the secretion of Nuc itself.

Endodeoxyribonucleases↗
PubMed1974

Genetic control of the immune response to staphylococcal nuclease. I. Ir-Nase: control of the antibody response to nuclease by the Ir region of the mouse H-2 complex.

A number of inbred and congenic resistant strains of mice were immunized with staphylococcal nuclease (Nase). Antibody responses were measured in the sera of the animals by a sensitive method involving inhibition of enzymatic hydrolysis of DNA, High responder strains included A/J, DBA/2, BALB/c, AKR/J, C57BR, and SJL/J. DBA/1 and C57BL/6 mice were low responders. The strain distribution of anti-Nase response potential was compatible with the relevant immune response gene(s) being linked to the murine major histocompatibility complex. Linkage of this response to H-2 was demonstrated by the findings that: (a) the congenic C3H/HeJ and C3H.SW mice were respectively high and low responders; (b) the congenic lines B10.A and B10.D2 were high responders, whereas the C57BL/10 strain was a poor responder; and (c) anti-Nase response potential of F(2) progeny from DBA/1 x SJL/J matings correlated with their H-2 type. Three B10.A recombinant lines were used to map this Ir gene within H-2. B10.A(4R) was a high responder to Nase, whereas B10.A(2R) and B10.A(5R) were both low responders. We wish to propose the name Ir-Nase for the gene(s) controlling antibody responsiveness to this immunogen. Our data indicate that Ir-Nase is located within the same chromosomal segment of the H-2 complex as is Ir-IgG.

Animals↗
PubMed1968

Plant nucleases. I. Separation and purification of two ribonucleases and one nuclease from corn.

Three enzymes with ribonuclease activity, one of which also had deoxyribonuclease activity, have been isolated and partially purified from corn seeds and seedlings. The purification of Ribonuclease I from mature seed was previously reported. This enzyme has a pH optimum near 5.0, is loosely adsorbed to carboxymethyl-cellulose, and has a molecular weight of 23,000, determined by gel filtration.Ribonuclease II was isolated from the microsomes of corn roots, and was partially purified by gel filtration. It has a pH optimum plateau from 5.4 to 7.0, and molecular weight of 17,000.Nuclease I hydrolyzes both RNA and DNA. It was isolated from the large particles of a corn root homogenate and was partially purified on a carboxymethyl-cellulose column. It has a pH optimum at 6.2 and a molecular weight of 31,000.The relative activities of the 3 enzymes for deoxyribonuclease and at pH 5 and pH 6.2 for ribonuclease may be used to characterize them during purification operations. Assays on homogenates of corn roots, and especially of the root tips, suggested that a fourth enzyme, which possesses deoxyribonuclease activity, is also present.

Journal Article↗
PubMed2005

Herpes simplex virus type 1 single-strand DNA binding protein ICP8 enhances the nuclease activity of the UL12 alkaline nuclease by increasing its processivity.

UL12 is a 5'- to 3'-exonuclease encoded by herpes simplex virus type 1 (HSV-1) which degrades single- and double-stranded DNA. UL12 and the single-strand DNA binding protein ICP8 mediate a strand exchange reaction. We found that ICP8 inhibited UL12 digestion of single-stranded DNA but stimulated digestion of double-stranded DNA threefold. The stimulatory effect of ICP8 was independent of a strand exchange reaction; furthermore, the effect was specific to ICP8, as it could not be reproduced by Escherichia coli single-stranded DNA binding protein. The effect of ICP8 on the rate of UL12 double-stranded DNA digestion is attributable to an increase in processivity in the presence of ICP8.

DNA↗
PubMed1996

The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli.

Staphylococcus aureus secretes two forms of extracellular nuclease, nuclease A and nuclease B. Nuclease A, consisting of 149 residues, is a proteolytic product of nuclease B, which is a processing intermediate that has a 19-residue N-terminal pro-peptide between the signal peptide and nuclease A. It has been shown that nuclease A can be secreted by Escherichia coli by fusing it to the OmpA signal peptide. We now demonstrate that the addition of the pro-peptide between the OmpA signal peptide and nuclease A leads to a significantly enhanced secretion rate in E. coli. The processing and secretion rates of nuclease B at 37 degrees C were at least 10 times faster than those of nuclease A. Nuclease B was also secreted efficiently under conditions which blocked the secretion of nuclease A, such as secA mutations and the addition of phenethyl alcohol or sodium azide. This enhancing effect of the pro-peptide was not as striking when it was attached to beta-lactamase, indicating that the pro-peptide acts as a specific secretion enhancer for nuclease A. Equilibrium circular dichroism on purified nuclease A and nuclease B indicated that the pro-peptide itself had no significant destabilizing effect on the mature protein. The existence of similar pro-peptides in Gram-positive bacterial secretory proteins indicates that they may also serve as secretion enhancers for individual proteins.

Adenosine Triphosphatases↗
PubMed1991

Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences.

Staphylococcus aureus nuclease A hybrid genes, encoding proteins OmpA-nuclease, lipo-nuclease and Pin-nuclease, were cloned downstream of the yeast GAL10 inducible promoter. OmpA-nuclease and lipo-nuclease contain the mature staphylococcal nuclease sequence preceded by the Escherichia coli OmpA and lipoprotein signal sequences, respectively, whereas Pin-nuclease lacks a defined signal sequence at its amino terminus. We found that: (a) the nuclease gene products synthesized in yeast are active, but they do not affect cell growth; (b) OmpA-nuclease and lipo-nuclease are partially processed and constitute approximately 1.0-1.5% of the yeast cell protein; (c) OmpA and lipoprotein signal sequences function similarly in secretion, allowing 35-40% of the processed nuclease to be translocated into the yeast periplasm; and (d) Pin-nuclease, which lacks hydrophobic sequences at its amino-terminus, is accumulated at a level tenfold lower than the hybrid proteins that do contain signal sequences. Nevertheless, 50% of the enzyme activity of Pin-nuclease in yeast is localized in the periplasmic space.

Amino Acid Sequence↗
PubMed1987

Inhibition of the catalytic properties of Staphylococcus aureus nuclease by monoclonal antibodies.

Monoclonal antibodies (Mab) specific for Staphylococcus aureus nuclease (nuclease) were examined for their capacity to inhibit the enzyme-mediated cleavage of DNA. Within a panel of 22 anti-nuclease Mab produced by hybridoma cell lines derived from SJL/J, A/J or BALB/c mice, only five were capable of modifying nuclease activity. Of the five, only one protected DNA from enzymatic degradation whereas the others reduced the rate of the enzymatic reaction. When mixed together, partially inactivating Mabs were frequently more efficient inhibitors than when used individually. It was shown by competitive binding assay that nuclease could be bound simultaneously to more than one Mab. Mixtures of five inactivating Mabs were able to completely block the nuclease activity. Although the actual mechanism for Mab nuclease inactivation is not known, the present data are consistent with simple steric hindrance for the formation of the DNA-nuclease complex by bulky Mab molecules bound to epitopes close to, but distinct from, nuclease catalytic sites. A mathematical model for Mab binding and inactivation of nuclease, taking into account multiple binding events for one or two Mabs interacting with nuclease, was used to derive affinities and maximum reductions of the enzymatic rate (details on the derivation of the equations and on the hypotheses of the model are given in an appendix). This analysis showed that the observed cooperative effects were dependent on the formation of multi-molecular complexes in which nuclease is bound simultaneously to two (or more) different Mabs. It also shows that the formation of cyclic complexes, if allowed, might result in very high apparent affinities. Since in screening of hybridoma fusions, the probability of finding such pairs of monoclonal antibodies would be low, this phenomenon may explain the fact that no Mab, or mixture of Mabs, matched the polyclonal antisera in capacity to block nuclease enzymatic activity.

Antibodies, Monoclonal↗
PubMed1981

Further study of the conformation of nuclease-(1-126) in relation to intrinsic enzymatic activity.

Nuclease-(1-126), although containing 89% of the amino acid sequence which folds to the ordered structure of nuclease A, is disordered and highly flexible (Taniuchi, H., and Anfinsen, C. B. (1969) J. Biol. Chem. 243, 4778-4786). On the other hand, Sachs et al. (Sachs, D. H., Schechter, A. N., Eastlake, A., and Anfinsen, C. B. (1974) Nature 251, 242-244) have demonstrated intrinsic enzymatic activity for nuclease-(1-126). To attempt to learn whether or not the active population of nuclease-(1-126) has the native conformation, we have examined nuclease-(1-126) with respect to enzymatic kinetics with and without the competitive inhibitor deoxythymidine 3',5'-diphosphate (pdTp), effect of temperature on enzymatic activity, binding of pdTp in the presence of Ca2+ and intrinsic viscosity, Stokes radius, CD, and response to trypsin action in the presence and absence of pdTp and Ca2+. The results indicate that the conformation of nuclease-(1-126) bound with pdTp in the presence of Ca2+ is partially constrained but still highly flexible below 30 degrees C, outside the range of thermal transition exhibited by the ordered elements of nuclease-(1-126). Thus, formation or stabilization of active site of nuclease-(1-126) by binding with ligands is not associated with cooperative folding of the entire polypeptide chain. Considering that nuclease-(1-126) does not bind to nuclease-(127-149) but does to nuclease-(111-149), the results are consistent with the idea that the specific cooperative interactions, providing extra stabilizing energy required for maintaining the polypeptide chain in the ordered state of nuclease A, may be disrupted for nuclease-(1-126) primarily due to cleavage of the peptide bond between residues 126 and 127. Then, it may be thought that binding with ligands does not compensate for this disruption.

Circular Dichroism↗
PubMed1990

Precursor-product relationship of larger to smaller molecular forms of the BAL 31 nuclease from Alteromonas espejiana: preferential removal of duplex exonuclease relative to endonuclease activity by proteolysis.

Two molecularly and kinetically distinct major species of the extracellular nuclease BAL 31 from Alteromonas espejiana, previously characterized as the "fast" (F) and "slow" (S) BAL 31 nucleases, have been evidenced to derive from proteolysis starting from a still larger (approximately 120 kDa) precursor nuclease. The expected protease activity in the culture fluid has been confirmed and is strongly dependent on the cell growth phase. The disappearance of the largest nuclease species with the concomitant sequential appearance of first the F and then the S species has been demonstrated for nuclease obtained from culture supernatants as a function of cell growth phase. Nuclease from periplasmic extracts displayed very little of the F and S nucleases. Treatment of purified F nuclease with Pronase or subtilisin readily converted it to species with only a few percent of the native exonuclease activity against duplex DNA but retaining much of the initial activity against single-stranded DNA. Electrophoresis in nuclease-detecting gels demonstrated a parallel conversion of the larger species to one indistinguishable in molecular weight from the S species. The observed loss of exonuclease activity could correspond to the conversion of the F to the S nuclease. However, treatment of S nuclease with subtilisin resulted in a drastic reduction of exonuclease activity of this enzyme on duplex DNA with retention of most of the activity against single-stranded and nicked circular duplex DNA substrates. Evidence of internal proteolysis of the S nuclease could be seen after electrophoresis in denaturing gels but only after the denaturation buffer was adjusted to 6 M in urea. The preferential removal of the exonuclease activity may enhance the usefulness of the BAL 31 nuclease in such applications as heteroduplex mapping.

Blotting, Western↗
PubMed1998

Purification, characterization and complete amino acid sequence of nuclease C1 from Cunninghamella echinulata var. echinulata.

It is known, from the zymogram method of nuclease activity assay, that the crude extracts of Cunninghamella echinulata var. echinulata contained at least three distinct extracellular nucleases. Among them, the major form was 30 kDa in molecular mass and termed nuclease C1. In this report, nuclease C1 was purified to apparent homogeneity by chromatography on Cibacron blue-3GA, phenyl-Sepharose 4B and HiTrap Heparin. Nuclease C1 acquired enzymatic activity in the presence of Mn2+ or Mg2+ and was inhibited by EDTA. The activity was maximal at pH 7-8.5. The primary structure of nuclease C1 was completely determined using enzymatic digestion and gene cloning. The N-terminal 49 residues of nuclease C1 were first elucidated from a tryptic digest. Two degenerate upstream primers were subsequently designed to amplify the cDNA encoding nuclease C1. The resulting protein sequence of nuclease C1 was shown to be composed of 252 residues. It was intriguing to find that the protein sequence of nuclease C1 showed significant similarities with the sequences of the mitochondrial nucleases of Saccharomyces cerevisiae (44% identity) and Schizosaccharomyces pombe (42% identity). Residue His87 of nuclease C1 was postulated to be located at the active site from sequence similarity with secreted nuclease from Serratia marcescens.

Amino Acid Sequence↗
PubMed1989

Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution.

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.

Calcium↗