Nuclease, extracellular

The leader sequence of the Serratia marcescens extracellular nuclease has been removed and the gene for the resulting nontransportable protein cloned behind the leftward promoter (PL) of lambda (Ahrenholtz, Lorenz Wackernagel, 1994). In the presence of a temperature-sensitive repressor (cl857), the cells can be induced to produce this altered enzyme by an increase in temperature. This produces the nuclease within the cell and, because it cannot be exported, its intracellular concentrations rise, rapidly degrading the cellular genome. Limitations to this system are that cell survival is reduced to only 2 x 10 5. 

In the second edition of this work the subject of the extracellular nuclease of Staphylococcus aureus, ribonucleate (deoxyribonucleate) 3 -nucleotidohydrolase, EC 3.1.4.7, was quite adequately covered in some six-hundred words (1). In this edition there are two chapters jointly containing more than an order of magnitude more words about this formerly obscure, nonspecific nuclease with its high pH optimum (pH 9-10) and... 

V8, as indicated by two-dimensional peptide maps of tryptic digests (44)- It appears, therefore, that various strains of S. aureus produce a major extracellular nuclease with covalent structures extremely similar to that of nuclease V8. Staphylococcus aureus has been reported, however, to produce several minor extracellular nucleases with different isoionic points and heat stabilities 45)- The covalent structures of these materials have not been studied. 

Benedik M. J. and Strych U. (1998) Serratia marcescens and its extracellular nuclease. LEMS Lett. 165(1), 1-13. 

Upon administration to the body, plasmid DNA or its complex with any vector, interacts with cells, proteins, and extracellular matrix. The summation of these interactions will determine the in vivo fate of plasmid DNA and its complex, which, in turn, determines the location and extent of gene transfer. From a pharmaceutical and biopharmaceutical point of view, plasmid DNA is a huge macromolecule with a strong negative charge, and it is susceptible to attack by nucleases. 

Serum contains proteins and nucleases that can interact with extracellular DNA and promote degradation. It has been demonstrated that in vitro transfection efficiency using most cationic lipids is reduced in the presence of fetal bovine serum (5-10%) (114). The stability of DNA in serum therefore indicates the degree of protection afforded to the DNA by the lipid carrier. 

In contrast to the lack of detailed structural information for the 3, 5 -cyclic nucleotide phosphodiesterase, staphylococcal (or micrococcal) nuclease (SNase), an extracellular nuclease produced by Staphylococcus aureus, is well characterized. SNase is an extraordinarily efficient catalyst for the endo- and exonucleo-lytic hydrolysis of single-stranded DNA and RNA, with the rate acceleration for DNA being approximately 10 relative to the uncatalyzed rate the final products of the reaction are 3 -mononucleotides. The sequence of 149 amino acids that constitute the enzyme has been determined both by classical degradation procedures and by sequence analysis of the cloned gene. A highly refined 1.65-A X-ray structure determined in the presence of Ca and the competitive inhibitor thymidine 3, 5 -bisphosphate (pdTp) was recently completed (87) this structure differs only slightly from a less refined 1.5-A structure that was reported in 1979... 

Several extracellular enzymes have one or more Ca + ions as integral parts of their structure. In a very few of them the Ca ion is bound at or near the active cleft, and appears necessary for maintaining the catalytic activity (phospholipase A2, a-amylase, nucleases), whereas other enzymes show catalytic activity even in the absence of Ca (trypsin and other serine proteases). In the latter proteins, the Ca + ion is usually ascribed a structural role, although its function may be rather more related to dynamics and so be more subtle and complex. 

In terms of structural chemistry, liposomes are supramolecular aggregates. A drug will also associate with the liposome by via non-covalent binding. With modified and unmodified ODNs, liposomes have become a particularly promising approach, aided by the fact that specific ODN-modifications may also be used specifically to influence drug-liposome interaction. Thus, base-modified cholesterol derivatives have been developed to act as specific anchor points for ODNs in liposomal formulations [397-408], Application of pH-sensitive liposomes made from DOPE/OA [409] or DOPE/OA/CHOL [410] has also been suggested. As a first important effect of liposome formulation, protection of ODNs from extracellular nucleases has been observed [411-413]. Use of such formulations has proven to be a valid method for cellular internalization in cultured cell lines, an effect which has been reviewed by a number of authors [414,415]. 

As we have described earlier, there are enzymes which are extracellular and are used in digestion. A considerable number of proteases, saccharrases, and nucleases are calcium-dependent. However, many other processes which are as complicated as digestion require calcium. Blood clotting, for example, is assisted by calcium-activated processes and may... 

Less than 3% of soil organic phosphorus is present as nucleic acids and derivatives derived from the decomposition of living organisms (Dalai, 1977). The four bases of DNA have been identified in humic acids (Anderson, 1961). The presence of nucleic acids and derivatives in the soil was confirmed by the isolation of two pyrimidine nucleoside diphosphates (Anderson, 1970). Nucleic acids are rapidly mineralized, re-synthesized and combined with other soil constituents, or incorporated into microbial biomass (Anderson and Malcolm, 1974). Nevertheless, the interaction of nucleases with soil constituents can inhibit DNA hydrolysis, with important environmental consequences related to extracellular gene uptake by bacteria (Demanfeche et al., 2001). 

No distinct structural motif is used by calcium-dependent nucleases such as staphylococcal nuclease.Since these enzymes are extracellular, where Ca + concentrations are high, the binding affinities are normally low and in the millimolar range (Table 1). 

---