DNase Research Articles

Purification and some properties of an acid deoxyribonuclease from testes of chinook salmon Oncorhynchus tshawytscha

An acid deoxyribonuclease, purified 5900-fold from aqueous extracts of salmon testes ( Oncorhynchus tshawytscha), was at least 10–15 times as potent as commercially purified hog spleen deoxyribonuclease II. The enzyme differs from an acid deoxyribonuclease isolated earlier from salmon testes, in that SO 4 2− was strongly inhibitory, while Mg 2+ was apparently not required for activity. The pH optimum was near 4.7. Heat-denatured DNA was hydrolyzed at approx. 10 % of the rate of native DNA. Poly d(A-T) alternating copolymer was hydrolyzed 1.5 times as rapidly as native DNA, but no appreciable hydrolysis of poly (dG) · poly (dC) homopolymer was observed. The initial products of DNA digestion consisted mainly of polynucleotides. Appreciable quantities of mononucleotides were also formed, particularly following prolonged digestion of DNA, thus suggesting the presence of some phosphodiesterase activity in the final preparation. The molecular weight of the testes enzyme was estimated by gel filtration to be approx. 38 000.

The DNase activities of the mouse.

AbstractSeveral tissues of the mouse contain neutral DNase activity, as well as the acid DNase found in lysosomes. The mouse pancreas was found to have very low levels of neutral DNase I activity, in contrast to the high levels of DNase I measurable in bovine pancreas.On the other hand, the mouse parotid salivary gland produces large amounts of DNase I which is secreted into the saliva.The parotid DNase can be distinguished from the neutral DNase found in the liver on the basis of acid‐ and heat‐stability, and from the mouse pancreas DNase I on the basis of acid‐stability.The mouse parotid DNase I is similar to the beef pancreas DNase I in certain of its properties, but shows a distinctly different band pattern on zonal electrophoresis.It is suggested that the parotid DNase I, like the pancreatic enzymes, is a cell‐specific molecule.

Neuraminidase activity in hela cells: effect of hydrocortisone.

Homogenates of HeLa cells contain neuraminidase activity. This enzyme is particle-bound, and it has a pH optimum of 4.2. Hydrocortisone-regulated cells contain two to three times as much neuraminidase as the corresponding controls. The hydrocortisone treatment also causes an increase in the cell content of beta-glucuronidase and acid deoxyribonuclease.

Studies on acid deoxyribonuclease: VII. Conformation of three nucleases in solution

The optical rotatory dispersion, circular dichroism, and infrared spectra of acid DNase, pancreatic DNase, and Staphylococcus aureus nuclease have been determined. The conformations of these three nucleases differ greatly from each other and from that of ribonuclease; while the acid and pancreatic DNases contain little α-helix, the bacterial enzyme has ca. 25% of that conformation; acid DNase probably contains antiparallel pleated sheet β-structure.

An Exonuclease Specific for Double Stranded Deoxyribonucleic Acid

Abstract A nuclease specific for double stranded DNA has been isolated and partially purified from Hemophilus influenzae. The enzyme has a pH optimum of 8.2 to 8.4 and requires a divalent cation, either Mg++ or Mn++. It is most active on DNA whose size has been reduced by either sonic disruption or limited digestion with pancreatic deoxyribonuclease or spleen acid DNase. The enzyme releases only 5'-mononucleotides and leaves a single stranded segment of DNA which is resistant to further hydrolysis. Furthermore, the enzyme destroys the transforming activity of H. influenzae DNA very slowly. These results indicate that the enzyme is an exonuclease, hydrolyzing from the ends of DNA molecules. In addition, the enzyme also exhibits a DNA-phosphatase activity and releases inorganic phosphate from DNA fragments which terminate in 3'-phosphates, suggesting that this enzyme begins hydrolysis from the 3' end of the DNA strands.

Periodic increase in deoxyribonuclease activity during the cell cycle in synchronized euglena.

Periodic increase in deoxyribonuclease activity during the cell cycle in synchronized euglena.

Conversion of Non-Immune Rabbit Spleen Cells by Ribonucleic Acid of Lymphoid Cells from an Immunized Rabbit to Produce IgG Antibody of Foreign Light Chain Allotype

Abstract Spleen cells (SpC) from non-immunized rabbits were converted to antibody “indirect” plaque-forming cells (PFC) by incubating them with ribonucleic acid (RNA) extracts of lymph node cells (LNC) obtained from rabbits 18 to 24 days after they were immunized with several intravenous injections of 4 × 108 sheep red blood cells (SRBC). The RNA extract was inactivated by ribonuclease (RNAse) but not by deoxyribonuclease (DNAse) or trypsin, indicating that the conversion required intact RNA. “Indirect” PFC were developed with optimal amounts of goat anti-IgG-Fc fragment and these were not inhibited by 2-mercaptoethanol (2-ME) or goat anti-IgM but were inhibited by excess anti-IgG-Fc fragment. This indicated that the antibodies produced by the PFC are of the IgG immunoglobulin class. The conversion was specific for the SRBC antigen injected. The light chain allotype of the IgG antibody produced by the PFC was identified by use of anti-b4 and anti-b5 to develop the “indirect” PFC, to visualize the PFC by radioautography and to inhibit plaque formation. Most of the “indirect” PFC of the converted SpC (64% to 84%) possessed IgG antibody with the allotype characteristic of the donor of the RNA extract; some of the “indirect” PFC (4% to 32%) possessed IgG antibody with the allotype characteristic of the donor of SpC. When the “RNA-converted” SpC were lysed by freezing and thawing, the anti-SRBC titer of the lysate, measured by the localized hemolysis in gel technique, was 4096 compared to 4 for lysates from the same number of lysed, nonimmune SpC alone. Moreover, anti-SRBC antibody could not be detected in the RNA extracts. These results indicate that a component of the RNA extract, presumably RNA itself, is providing information for the synthesis of at least part of the IgG antibody molecule.

Digestive Enzymes of the American Lobster (Homarus americanus)

Gastric juice of lobster hydrolyzed the following substrates: triolein (enzymic activity: lipase), tributyrin ("lipase"), Azocoll(R) (proteinase), TAME and BAEE ("trypsin"), ATEE ("chymotrypsin"), HPLA ("carboxypeptidase A"), DNA (deoxyribonuclease), RNA (ribonuclease), p-nitrophenyl-phosphate (phosphatase), and p-nitrophenyl-N-acetyl-β-glucosaminide (chitobiase). (The quotation marks signify that the substrate specificities are typical of the quoted enzymes of mammalian or microbial origin. The spectra of specificities, however, of these enzymes and the corresponding enzymes of lobster are not necessarily identical.) Very low activities were found against RBB-starch (amylase) p-nitro-phenyl-α-(and β)-glucopyranoside(α- and β-glucosidase), p-nitrophenyl-β-galactopyranoside (β-galactosidase), and chitin azure (chitinase). No activities corresponding to phospholipase (lecithin), carboxypeptidase B (benzoylglycyl-L-arginine), elastase, glycylglycine dipeptidase, or leucine aminopeptidase were found.Gel filtration of gastric juice proteins on Sephadex(R) G-100 yielded estimates of molecular weights, among them: chitobiase 100,000; phosphatase 80,000; lipase 43,000; DNase 33,000; "trypsin" and ribonuclease 25,000; and two proteinases with optima around pH 4 and 8 and the low molecular weight of 12,500; proteolytic activity at pH 4 was also associated with the molecular weights 25,000 and 50,000.In ion-exchange chromatography, enzymes were eluted from DEAE-cellulose in the following sequence of increasing anionic character: chitobiase, proteinase (pH 4), proteinase (pH 8), lipase, "carboxypeptidase A," DNase, phosphatase, and "trypsin" and "chymotrypsin."Of the principal enzymes, only one (chitobiase) had its optimum at the pH 5 normal for lobster gastric juice. The lipase had an optimal pH around 7, phosphatase near 9, and "trypsin" near 8; proteolytic activity shows a maximum at pH 7–8 and another maximum, unusual for Crustacea, at pH 4.Much of the protein of gastric juice could not be connected with any enzymic activity. The proportion of nonactive protein seemed to increase with starvation of the animals. We found no evidence for the occurrence of zymogens.

The effects of alkylating agents and DNA ase on nucleic acid synthesis in rat-liver nuclei in vitro

Adult female rat-liver nuclei have been treated with nitrogen mustard (HN 2), methylene dimethane sulphonate (MDMS) and bovine pancreatic DNAase I. DNA synthesis and RNA synthesis in this system have been assayed using the methods of labelled TTP and UTP uptake. The results indicate that low concentrations of MDMS and HN 2 stimulate RNA synthesis and that this effect may be simulated by treatment with low levels of DNAase I. However, a similar parallelism between the actions of higher concentrations of MDMS and DNAase does not appear to exist since at such concentrations MDMS does not affect DNA synthesis whereas DNAase stimulates DNA synthesis. At these levels, however, both MDMS and DNAase inhibit RNA synthesis. It is suggested that low levels of MDMS and HN 2 affect RNA synthesis in rat-liver nuclei by inducing breakage of the DNA template, whereas higher levels of these agents could inhibit RNA synthesis in the nuclei by a different mechanism.

Studies on chloroplast development and replication in Euglena: II. Identification of two different deoxyribonucleases

Extracts of Euglena contain at least two DNases. One, referred to as “Euglena acid DNase,” has a pH optimum of 4.6, and exhibits enzymic and purification properties similar to DNase II-like enzymes. The second DNase, referred to as “Euglena alkaline DNase,” has a pH optimum of 9.4, requires Ca 2+, and acts preferentially on heat-denatured DNA. Euglena alkaline DNase is either absent, present in low activity, or inhibited in dark-adapted cells; in addition, the activity of this DNase increases during light-induction of chloroplast development. We hypothesize that Euglena alkaline DNase is a chloroplast-related enzyme.

Secretion from the bovine adrenal gland: Release of lysosomal enzymes

Isolated bovine adrenal glands were perfused with a physiological salt solution and exposed to various secretogogues. Perfusates were assayed for catecholamines, protein, lactate dehydrogenase, acid ribonuclease, acid deoxyribonuclease, cathepsin, acid phosphatase and β-glucuronidase. Stimulation of the gland with carbachol induced secretion of catecholamines, protein and acid hydrolase activity. Perfusate LDH activity was unchanged after carbachol stimulation. Carbachol stimulation of the perfused isolated medulla evoked deoxyribonuclease secretion; the relationship between catecholamine secretion and secretion of deoxyribonuclease activity was the same for the perfused whole gland and isolated medulla. Secretion of deoxyribonuclease activity was also evoked by acetylcholine, nicotine, potassium chloride and Triton X-100, agents that also cause catecholamine secretion. Carbachol-induced secretion of deoxyribonuclease activity was reduced in the presence of tetracaine or during perfusion with calcium-free fluid. The introduction of calcium chloride during perfusion with calcium-free fluid also caused secretion of deoxyribo-nuclease activity. It is concluded from these results that stimulation with carbachol of the isolated perfused bovine adrenal gland causes secretion of deoxyribonuclease activity from the adrenal medulla, and that the secretion process is dependent upon the presence of calcium.

Lysosomal enzymes in the bovine adrenal gland: A comparison of medulla and cortex

Acid ribonuclease, acid deoxyribonuclease, cathepsin, acid phosphatase and β-glucuronidase activity were measured in 0.3 M sucrose homogenates of bovine adrenal medulla and cortex. Significant amounts of each of these enzymes were associated with the mitochondrial fractions of each tissue. Sucrose density gradient centrifugation of the mitochondrial fractions showed that the acid hydrolases were present in particles distinct from mitochondria and, in medulla, distinct from chromaffin granules. Each of the hydrolases exhibited an acid pH optimum. Acid hydrolase activity of homogenates and of density gradient fractions was increased after several freeze-thaw cycles, upon incubation at 50° for 10 min, upon incubation at 37°, pH 9.0, for 15 min, after exposure to 0.1% Triton X-100, and with sonication for 60 sec. It was concluded from these data that in both cortex and medulla the acid hydrolases are associated with a population of subcellular organelles that possess the characteristics of lysosomes.

Studies on the nonspecific phosphodiesterase activity of splenic acid deoxyribonuclease

Studies on the nonspecific phosphodiesterase activity of splenic acid deoxyribonuclease

Effect of glucocorticoids on the acid DNase of the thymus.

The acid DNase of the rat thymus was shown to have characteristics similar to the DNase of other tissues. It had a pH optimum of 5.3 in the absence of added divalent cations; MgCl at 0.05m activated the enzyme and reduced the pH optimum to 4.4. Cortisol treatment for 4 hr increased the total activity as well as the specific activity of this enzyme in the thymus. The response to cortisol occurred in both adrenalectomized and intact rats. The measured increase in acid DNase was in the range of 20 to 30% when the thymus was homogenized in glass distilled water, and 30 to 50 % when the homogenization medium was 0.25m sucrose plus 0.1m CaCl2. The mechanism for this increase in activity of DNase was investigated. Actinomycin, cycloheximide and puromycin had little effect on the cortisol-induced increase. No evidence was obtained that the elevated activity resulted from a release of enzyme from a subcellular particle, nor did the data support the idea of increased specific activity as a result of selective prote...

Studies on acid deoxyribonuclease from brain.

An endonucleolytic acid DNase was purified from beef brain. It resembled spleen DNase II with respect to the properties studied: preference for double-strandedness, products of hydrolysis, pH optima, effects of some salts, and the effect of high substrate concentrations on the rate of release of acid-soluble nucleotides. The latter three points are discussed.

A protein inhibitor of acid deoxyribonucleases.

A protein extracted and partially purified (about 100-fold) from mouse liver is able to inhibit the acid DNases from different tissues and species, whereas pancreatic DNase and E. coli endonuclease I are not inhibited. The acid DNase displays typical Michaelis-Menten kinetics in the absence of this inhibitor, but the kinetics become sigmoidal in its presence. The existence of a DNase-inhibitor complex is demonstrated by physicochemical experiments. Moreover, the inhibitor is able to reactivate the DNase treated by urea, probably through a reassociation of the inactive monomers to a dimeric state. An allosteric model in which the DNase-inhibitor complex is composed of catalytic (DNase) and regulatory (inhibitor) subunits could explain these data.

Secretion from the cortex-free bovine adrenal medulla.

1. The isolated bovine adrenal medulla was prepared from whole glands by careful removal of the cortex. The tissue was perfused with physiological salt solution and stimulated with a variety of secretogogues.2. Catecholamines were secreted from the tissue upon stimulation with carbamylcholine, nicotine sulphate, acetylcholine bromide, histamine dihydrochloride, (+)-amphetamine sulphate and potassium chloride.3. Carbachol-induced secretion of catecholamines was reduced in the presence of either hexamethonium bromide or tetracaine hydrochloride, during perfusion with calcium-free perfusion fluid, or during perfusion at low temperature.4. Stimulation of the medulla with carbachol also led to secretion of chromaffin granule protein and acid deoxyribonuclease. The relationships between catecholamines secreted and the amounts of these proteins secreted were similar to the corresponding values for the perfused whole bovine adrenal gland. Perfusate lactate dehydrogenase activity and perfusate haemoglobin content were unchanged after carbachol stimulation.5. It is concluded that there are no differences in the mechanisms for catecholamine secretion from the cortex-free perfused medulla and the perfused whole gland.

Studies on pulmonary secretions. 3. The nucleic acids in whole pulmonary secretions from patients with cystic fibrosis, bronchiectasis, and laryngectomy.

The presence of deoxyribonucleic acid (DNA) in the pulmonary secretions of patients with cystic fibrosis, bronchiectasis, and laryngectomy has been confirmed by direct isolation and chemical characterization. No abnormal bases have been detected in the isolated DNA, and the base composition is closely related to that of normal human DNA. The DNA is contained within the water-insoluble fraction of the secretions. Ribonucleic acid has not been demonstrated by a variety of chemical and enzymic tests. The isolated DNA from whole secretions or the water-insoluble fraction is readily split by exogenous deoxyribonuclease (DN ase I or streptodornase). The viscosity of the secretions shows an apparent decrease concomitant with the enzymic splitting. At pH 7, exogenous DNase II shows less, but detectable, activity against the DNA of the secretions, and this activity was enhanced by adjustment of the pH to 5. Endogenous DN ase activity could not be demonstrated on incubation of fresh pulmonary secretions. The DNase activity of cystic fibrosis tissues is comparable both qualitatively and quantitatively to that of normal control tissues. The activity of the nucleases liberated into the secretions may be impaired by an unfavorable pH for the action of DNase II and the presence of a naturally occurring inhibitor for DNase I. The accumulation of DNA in pulmonary secretions is probably a result of the exudative inflammatory process outstripping the disposal mechanisms of the lung.

Insoluble matrix-supported apyrase, deoxyribonuclease and cholinesterase

Insoluble matrix-supported apyrase, deoxyribonuclease and cholinesterase

Correlation of nucleolytic activities with the stage of growth of the silkworm

From the tissues of the silkworm several nucleolytic enzymes were detected. Among these, acid ribonuclease (RNase) and alkaline nuclease were dominant. The optimal pH values for these are 5·3 and 10·3, respectively. An acid deoxyribonuclease (DNase), optimally active at pH 5, was also found in the tissues of the insect. Changes of activity were investigated of the acid RNase and the alkaline nuclease through the whole life cycle of the silkworm. The former remains very active during each step of development. By contrast, the latter exists at higher levels only during the feeding period, and then drops abruptly to an almost undetectable level at the initiation of spinning. The acid DNase activity showed a peak 3 days after pupation. The acid RNase and DNase showed a wide distribution in the tissues of the silkworm, whereas the alkaline nuclease is restricted to the gut juice and to the gut.