PubMed Health⌕ Search

PubMed · 2628267

A sensitive assay for Staphylococcus aureus nucleases.

Abstract

A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured [3H]thymidine labelled DNA from E. coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

J K Kohli, B V Vakil, M S Patil, V N Pandey, D S Pradhan. 1989. A sensitive assay for Staphylococcus aureus nucleases.. https://pubmed.ncbi.nlm.nih.gov/2628267/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

RNases and nucleases in embryos and endosperms from naturally aged wheat seeds stored in different conditions.

Temperature and moisture content are particularly important factors influencing the longevity of seeds, and therefore the ageing of seeds is closely tied to storage conditions. The ageing process is characterised by many physiological and biochemical changes: membranes tend to leak, enzymes lose catalytic activity, and chromosomes accumulate mutations. Since viability loss is also associated with the breakdown of nucleic acids, the aim of the study was to determine whether the damage induced by ageing could be associated with changes in the activity of RNases and nucleases in embryos and endosperms of differently stored wheat seeds. In order to better characterise seed conditions, the damage to membranes during seed ageing was evaluated by measuring the conductivity of the soaking solution during imbibition, and by using the Evans Blue colorant; lipid peroxidation was also recorded. RNases and nucleases were studied by SDS-PAGE and activity staining. Ageing of seeds stored in a dry state involved a progressive loss of membrane integrity, which increased with the degree of ageing, while lipid peroxidation remained unchanged. Changes in nucleolytic enzyme activity were recorded in embryos: a decrease in RNases and an increase in nucleases. In the endosperm compartment there were no significant differences in ribonuclease and nuclease patterns during seed ageing. Moreover, neutral RNases were absent in endosperms of dry seeds and were activated following imbibition. Present studies reveal that embryos and endosperms have different enzymatic patterns, thus highlighting that the two seed compartments age independently. A different nucleolytic pattern was present in seeds of comparable viability and membrane damage, which were stored differently, and nuclease metabolism was subject to regulation according to both ageing and the length of the storage period.

Deoxyribonucleases↗

Macrocyclic lanthanide complexes as artificial nucleases and ribonucleases: effects of pH, metal ionic radii, number of coordinated water molecules, charge, and concentrations of the metal complexes.

We have been interested in the design, synthesis, and characterization of artificial nucleases and ribonucleases by employing macrocyclic lanthanide complexes because their high thermodynamic stability, low kinetic lability, high coordination number, and charge density (Lewis acidity) allow more design flexibility and stability. In this paper, we report the study of the use of the europium(III) complex, EuDO2A+ (DO2A is 1,7-dicarboxymethyl-1,4,7,10-tetraazacyclododecane) and other lanthanide complexes (i.e., LaDO2A+, YbDO2A+, EuK21DA+, EuEDDA+, and EuHEDTA where K21DA is 1,7-diaza-4,10,13-trioxacyclopentadecane-N,N'-diacetic acid, EDDA is ethylenediamine-N,N'-diacetic acid, and HEDTA is N-hydroxyethyl-ethylenediamine-N,N',N'-triacetic acid), as potential catalysts for the hydrolysis of the phosphodiester bond of BNPP (sodium bis(4-nitrophenyl)-phosphate). For the pH range 7.0-11.0 studied, EuDO2A+ promotes BNPP hydrolysis with the quickest rates among LaDO2A+, EuDO2A+, and YbDO2A+. This indicates that charge density is not the only factor affecting the reaction rates. Among the four complexes, EuDO2A+, EuK21DA+, EuEDDA+, and EuHEDTA, with their respective number of inner-sphere coordinated water molecules three, two, five, and three, EuEDDA+, with the greatest number of inner-sphere coordinated water molecules and a positive charge, promotes BNPP hydrolysis more efficiently at pH below 8.4, and the observed rate trend is EuEDDA+ > EuDO2A+ > EuK21DA+ > EuHEDTA. At pH > 8.4, the EuEDDA+ solution becomes misty and precipitates form. At pH 11.0, the hydrolysis rate of BNPP in the presence of EuDO2A+ is 100 times faster than that of EuHEDTA, presumably because the positively charged EuDO2A+ is more favorable for binding with the negatively charged phosphodiester compounds. The logarithmic hydrolysis constants (pKh) were determined, and are reported in the parentheses, by fitting the kinetic k(obs) data vs pH for EuDO2A+ (8.4), LaDO2A+ (8.4), YbDO2A+ (9.4), EuK21DA+ (7.8), EuEDDA+ (9.0), and EuHEDTA (10.1). The preliminary rate constant-[EuDO2A+] data at pH 9.35 were fitted to a monomer-dimer reaction model, and the dimer rate constant is 400 times greater than that of the monomer. The fact that YbDO2A+ catalyzes BNPP less effectively than EuDO2A+ is tentatively explained by the formation of an inactive dimer, [Yb(DO2A)(OH)]2, with no coordination unsaturation for BNPP substrate binding.

Deoxyribonucleases↗

Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation.

Aggregation of recombinant AAV2 results in reduced yield during purification and may have deleterious effects on vector transduction efficiency, biodistribution and immunogenicity following in vivo administration. Studies to elucidate the mechanism of vector aggregation and methods to prevent its occurrence are reported. In excipient screening studies, the sugars sorbitol, sucrose, mannitol, trehalose, or glycerol at concentrations of up to 5% (w/v), or surfactants Tween 80 or Pluronic F68, did not prevent aggregation. Aggregation was prevented by the use of various salts at concentrations corresponding to solution ionic strengths of >200 mM. AAV2 vectors purified by double cesium chloride gradient centrifugation, cation-exchange chromatography, or combined chromatography and gradient centrifugation each demonstrated a similar requirement for ionic strength to prevent aggregation. AAV2 vectors concentrated to 6.7 x 10(13) vector genome (vg)/mL in neutral-buffered isotonic saline resulted in 59+/-6.0% recovery of nonaggregated material compared to 96+/-4.4% recovery in an isotonic formulation with elevated ionic strength. The latter showed no aggregation following storage or after 10 freeze-thaw cycles at -20 degrees C. AAV2 vectors stored for an extended period in an elevated ionic strength formulation retained a high infectivity titer (13 vg/infectious unit) and transduction efficiency. Nuclease digestion of purified AAV2 vectors reduced aggregation, implicating trace amounts of vector surface nucleic acids in interparticle binding.

Deoxyribonucleases↗