DNase Research Articles

Absorption and secretion in the cauda epididymidis of the rabbit and the effects of degenerating spermatozoa on epididymal plasma after castration.

SUMMARY The origins of the constituents of epididymal plasma from the cauda epididymidis of the rabbit have been investigated. Results show that the epididymis actively absorbs Na+ and Cl− ions and actively secretes glycerylphosphorylcholine, protein, alkaline phosphatase, acid phosphatase, β-N-acetylglucosaminidase, α-mannosidase and acid deoxyribonuclease. Degenerating spermatozoa contribute K+ ions and material giving a positive reaction with diphenylamine reagent. It is suggested that in a normal animal, spermatozoa are suspended in a 'degradative milieu' and that this is involved in the disappearance of spermatozoa after castration.

Rapid analysis of 3′-ends of DNA fragments by terminal 32P-labeling

A fast method of analysis of the 3′ ends of oligodeoxyribonucleotides is described. Basically the method involves: (a) Labeling of the 3′ ends of oligodeoxyribonucleotides with the terminal deoxynucleotidyl transferase and [α- 32P] ATP as donor; (b) hydrolysis of the labeled fragments to 3′ deoxymononucleotides by acid DNase and spleen exonuclease; (c) unidimensional separation on polyethylene imine cellulose thin-layer plates of the four 3′ deoxyribomononucleotides, 3′ riboadenylic acid, and ATP.

Release of cytoplasmic enzymes into culture fluid.

AbstractThe release of enzymatic activities from cells grown in protein‐and lipid‐free synthetic media into culture fluids was investigated. Cell strains employed were the derivatives from mouse fibroblasts, rat liver parenchymal cells, rat ascites hepatoma cells and HeLa cells. Activities of acid DNase, acid RNase and alkaline phosphatase (ALP)‐I were detected in culture fluids as early as one day after renewal of medium, whereas those of β‐glucuronidase and acid phosphatase were not found. This release of enzymes was unlikely to be caused by cell disruption during cultivation.The release of Dnase was inhibited by the addition of cycloheximide or actinbomycin D, whereas that of ALP‐I was not inhibited.

The Effect of Internally Deposited Plutonium-239 on the Lysosomes of Rat Liver

The effects of plutonium-239, an α-particle emitting radionuclide which deposits mainly in the lysosomes, on the activity of various lysosomal enzymes have been studied in rat liver. The effects observed have been compared with those produced by the β + γ radiation from colloidal gold-198. Following administration of 0.5 μCi polymeric239 Pu the specific activities of acid deoxyribonuclease and aryl sulphatase were increased by 30 days and remained elevated at 1 yr. β-Glucuronidase, acid β-glycerophosphatase, and cathepsin D showed increased specific acitivity at time intervals longer than 6 mo after injection of239 Pu. All the enzymes studied showed decreased sedimentability at 30 days but this returned to approximately normal limits at longer time intervals. In comparison the administration of 0.5 mCi198 Au colloid also caused increased specific activity of deoxyribonuclease II, β-glucuronidase, and acid β-glycerophosphatase, but did not decrease the enzyme sedimentabilities. It is concluded that at leas...

Purification and Properties of Acid Deoxyribonucleases of Human Gastric Mucosa and Cervix Uteri

Abstract The acid deoxyribonucleases in homogenates of human gastric mucosa and cervix uteri were purified extensively by a procedure including DEAE-cellulose and phosphocellulose chromatographies, gel filtration, and isoelectric focusing. The enzymes in the two preparations had similar properties. The active protein was resolved into at least two forms with different isoelectric points, as judged by the formation of acid-soluble fragments. The major component of the activity had an isoelectric point at pH 7.02, and the minor component at pH 6.86. However, other properties of these two forms were similar. The final preparations of the gastric and uterine acid DNases were free of other types of DNase (DNases I, III, and IV), acid and alkaline phosphatases, acid and basic RNases, and nonspecific phosphodiesterase. Both enzymes had a molecular weight of approximately 38,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 5.2 to 5.5 in 33 mm acetate buffer. They did not require divalent cations for activity, and hydrolyzed native, double-stranded DNA about 10 to 20 times faster than heat-denatured DNA. They did not act on RNA or calcium bis(p-nitrophenyl) phosphate, indicating that they did not contain intrinsic phosphodiesterase activity. The products formed from native DNA were 3'-phosphoryl- and 5'-hydroxyl-terminated oligonucleotides. The average chain length of the limit digests with these enzymes was approximately 10 and the predominant species were longer than hexanucleotides.

Effect of Exogenous DNA on Acid Deoxyribonuclease Activity in Intact Roots of Vicia faba L.

Effect of Exogenous DNA on Acid Deoxyribonuclease Activity in Intact Roots of Vicia faba L.

Mode of Action of Acid Deoxyribonucleases from Human Gastric Mucosa and Cervix Uteri

The modes of endonucleolytic action of acid deoxyribonucleases (DNases) purified from human gastric mucosa and cervix uteri were investigated. The sedimentation patterns in alkaline and neutral sucrose gradients of digests of linear duplex DNA at an early stage of hydrolysis suggested that gastric DNase makes double strand scissions, while the uterine enzyme makes single strand scissions. To examine the initial kinetics of hydrolysis of the substrate molecule in more detail, λdv twisted circular duplex DNA was used as substrate, and the hydrolysis products were treated with ATP-dependent DNase, which acts specifically on double-stranded DNA in the linear form. The resulting products were sequentially analyzed by sucrose density gradient sedimentation. In this way open circular DNA and linear DNA formed from twisted, circular duplex DNA could be distinguished clearly. Using this method it was found that gastric DNase produces both open circular DNA and linear DNA simultaneously at a very early stage of hydrolysis, while the uterine enzyme produces open circular DNA first, and linear DNA after a lag phase. At 0° the gastric enzyme converted twisted, circular duplex DNA to the open circular form, introducing single strand scissions. These results indicate that both enzymes attack duplex DNA substrate molecules by making single strand scissions, but that the gastric enzyme makes single strand breaks first, followed immediately by second single strand scissions, resulting in the rapid formation of double strand cleavages.

A comparison of the biochemical changes induced in mouse brain by cuprizone toxicity and by scrapie infection

Studies were made of the activity of acid deoxyribonuclease and eleven glycoside hydrolases, and the rate of incorporation of DNA and glycoprotein precursors in the brains of mice fed on a diet containing 0·5 per cent. cuprizone (biscyclohexanoneoxaldihydrazone) for up to 16 weeks. The profile of changes observed was very similar to that found in the brains of mice infected with the Chandler strain of scrapie agent. The biochemical lesions of cuprizone toxicity in brain could be reversed by withdrawing cuprizone from the diet. It is concluded that many of the biochemical changes seen in Chandler scrapie are not specific for the disease and that cuprizone toxicity may be a useful control for certain types of scrapie experiment.

Purification of an acidic deoxyribonuclease II from the primate spleen

1. 1. Acid DNase from monkey and baboon spleens was isolated and purified by extraction at pH 3–4, (NH 4 2SO 4 fractionation, heating at 50° C for I hour, SP-C 50 chromatography and Sephadex G-100 gel chromatography. The final yield was 22 per cent. 2. 2. The results of polyacrylamide disc gel electrophoresis, isoelectrofocusing and analytical ultracentrifugation indicated that the preparations were pure and homogeneous. 3. 3. The optimum pH for the hydrolysis of highly polymerized DNA by the two acid DNases is 5.2. 4. 4. The two enzymes had essentially the same sedimentation coefficients (s 20 0, w, 2.5 × 10 1 ̄ 3 second) , diffusion coefficients ( D 20,w, 8 × 10 −7 cm 2. second −1), partial specific volumes ( v, 0.73 cm. 3 g. −1) , molecular weights (mol. wt. approximately 38,000), and isoelectric points (pI, 6.2).

The in vivo and in vitro effects of aflatoxin B1 on rat liver and spleen acid deoxyribonuclease

1. 1. The in vivo experiments with aflatoxin B 1 on rat liver acid DNase showed that the specific activity of the enzyme extract was dramatically increased. 2. 2. In contrast, the in vitro experiments revealed no direct activation effect of the toxin on acid DNase. 3. 3. The results of polyacrylamide disc gel electrophoresis and electrofocusing indicated that the acid DNase preparation from rat spleen was pure and homogeneous. 4. 4. The molecular weight and isoelectric point were calculated to be 36,000 and 10 respectively.

Fibroma virus-induced acid deoxyribonuclease in rabbit kidney cells.

Acid deoxyribonuclease (DNase) activity increased 6 to 10 fold in primary rabbit kidney (PRK) cells infected with Shope rabbit fibroma virus. Increase in enzyme synthesis was proportional to viral DNA replication. Addition of actinomycin D or puromycin to cultures terminated enzyme synthesis, indicating requirement for messenger RNA andde novo protein synthesis. The enzyme which was concentrated by ammonium sulfate precipitation and purified by DEAE cellulose chromatography degraded single and double-stranded DNA in a ratio of 2∶1 and required Mg++ ions for optimal activity. It was inactivated by more than 80 per cent in 5 minutes at 50° C and the presence of deoxynucleoside triphosphates in a reaction mixture inhibited its activity.

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL.

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in α-glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.

A new approach to the study of nucleotide sequences in DNAs.

The composition of the 3'terminal, 5'terminal and 5'penultimate nucleotides of the oligonucleotides released by spleen acid DNase and snail acid DNase from five ;repetitive' DNAs (guinea pig, mouse and crab satellite DNAs, yeast mitochondrial DNA and poly (dAT:dAT)) and four ;eukaryotic' DNAs (calf thymus, guinea pig and mouse liver DNAs, and yeast nuclear DNA) have been investigated and found to deviate in characteristic ways from those expected for bacterial DNAs having comparable base compositions. The deviation patterns obtained represent a novel way of characterizing and comparing different DNAs on the basis of the frequency of the nucleotide sequences they contain.

Lung Concentric Laminar Organelle: HYDROLASE ACTIVITY AND COMPOSITIONAL ANALYSIS

An analytical study was made of the concentric lamellar organelles (CLO) isolated from rabbit lung. These subcellular structures, which are normally located in the type II cells of mammalian lungs, were purified by flotation using a discontinuous sucrose density gradient. Electron microscopy of the isolated CLO revealed many intact structures about 1 µm in diameter with densely packed trilaminar membranes having, also, a granular core and an outer limiting membrane. The CLO contained a preponderance of polar lipids, mostly as phosphatidylcholine and phosphatidylethanolamine, with trace amounts of neutral lipid. A phospholipid to protein ratio of approximately 12:1, was repeatedly obtained and served as a criterion for organelle purification. Acrylamide gel electrophoresis of the detergent-solubilized CLO revealed a prominent staining band migrating with albumin. These structures had numerous hydrolases similar to those observed in lysosomes. High specific activities were observed for such enzymes as acid phosphatase, aryl sulfatase and β-N-acetylglucosaminidase. Multiple forms of the latter enzyme were observed in zonal electrophoresis studies. Purification of the CLO resulted in a 20- to 40-fold increase in specific activity over homogenates for most of the hydrolases. Cathepsin D, acid DNase, acid RNase and neuraminidase, other lysosome-associated hydrolases, were not detected in the CLO fraction. It is suggested from these and other studies that CLO are secreted to the acellular lining of lung where they contribute surfactant phospholipids, and possibly, functional hydrolases. The lung CLO also appear to have many properties in common with those subcellular structures associated with certain human genetic lipidoses.

Gastric carcinogenesis in rat induced by methylnitrosourea (MNU). Morphology, and histochemistry of nucleases.

27 squamous cell carcinomas of the forestomach, 2 adenocarcinomas of the glandular stomach and very numerous preneoplastic lesions of the mucosa in both parts of the stomach were induced after chronic oral administration of MNU in Wistar R rats. Single transplacental administration of MNU did not produce any gastric tumor, however preneoplastic alterations were detected in the forestomach of 42 animals and in the glandular stomach in 25 animals out of 51 examined, showing histologic patterns like those found in rats after chronic oral MNU administration. The pre- and neoplastic alterations in the forestomach demonstrated a distinct decrease of acid and alkaline DNase and RNase activity as compared with the normal forestomach. The last mentioned observation and our previously published results suggest that the deficiency of nucleases precedes or facilitates carcinogenesis.

Isolation and transcription of 3' terminal regions of unique DNA molecules.

ONE of the major difficulties in the determination of the primary structure of DNA is the isolation of small specific polydeoxyribonucleotide fragments which can be analysed directly or transcribed into RNA. So far, this difficulty has been overcome only in the case of regions that can be selectively protected from endonucleolytic attack by binding to highly purified specific proteins or to ribosomes. We have developed a method which allows the selective isolation and subsequent transcription of fragments derived from the 3′ termini of any unique DNA molecule. The procedures were established by using the DNA from bacteriophage λ; this provided an indication of the validity of the technique for relatively high molecular weight DNA. The method involves the limited digestion of the DNA with spleen acid deoxyribonuclease to produce fragments of chosen length, the selective extension of the fragments derived from the 3′ termini of the original DNA by addition of dATP residues with terminal deoxynucleotidyl transferase and the separation of the extended fragments from the bulk of the DNA by chromatography on oligo (dT) cellulose (Fig. 1). The RNA transcripts prepared from these fragments exhibited an absolute specificity for the 3′ terminal regions of A DNA in a variety of hybridisation experiments. Two-dimensional fingerprint analysis of ribonuclease T1 digests of the transcription products gave a simple and characteristic oligonucleotide pattern which could be used for sequence determination. This method can also be adapted to the isolation of specific fragments from internal regions of DNA molecules.

Studies on the Specificity of an Acid Deoxyribonuclease from Helix aspersa (Müll.)

The kinetics of degradation of calf thymus DNA by the DNAase from Helix aspersa has been investigated in its middle and terminal phase by determining both hyperchromic shift and average chain length of the digest. The results obtained are practically identical with those previously reported for acid DNAase from hog spleen. In contrast, the distribution of the isostichs in snail DNAase digests is significantly different from those found for spleen acid DNAase digests of similar sizes.The 3′‐phosphate and 5′‐hydroxy terminal nucleotides were found not to vary in their compositions in the average size range 34 to 10 and 70 to 12, respectively. In the 3′‐phosphate terminal position, deoxyadenosine is by far predominant (78%) and deoxycytidine almost absent (less than 1%); deoxyguanosine and thymidine form 6% and 16%, respectively, of the terminals. In the 5′‐hydroxy terminal position deoxyguanosine is predominant (45%), followed by deoxycytidine (31%), thymidine (14%) and deoxyadenosine (10%); in the 5′‐hydroxy penultimate position thymidine forms 38% of the nucleotides, deoxyguanosine 24%, deoxyadenosine 21% and deoxycytidine 17%.

Further Investigations of the Specificity of an Acid Deoxyribonuclease from Helix aspersa (Müll.)

Linear relationships were found to hold between the percentages of the 3′‐P terminal and 5′‐OH terminal and penultimate nucleotides released by snail acid DNAase from bacterial DNAs and the (dG + dC) contents of the latter. This finding is similar to previous results obtained on spleen acid DNAase; as in the case of this enzyme, the composition of the termini released by the snail DNAase from calf thymus DNA devite from the linear relationships mentioned above.The snail enzyme appears to recognize a sequence of at least two nucleotides, namely those which will be released as 3′‐P and 5′‐OH terminlas, respectively.

Effects of chronic ethanol treatment on rat liver lysosomes.

Abstract: The subcellular distribution and the latency of the lysosomal enzymes b‐glucuronidase and acid DNase have been studied in livers from ethanol treated and control rats. Some livers were isolated and perfused with or without ethanol. Ethanol treatment (4‐5 weeks) increased the free (non‐latent) lysosomal enzyme activity significantly without affecting the total activities. In the perfused liver ethanol produced a small stabilizing effect on the lysosomes. The lipid content of the liver did not change, and the plasma enzymes (alanine aminotransferase, aspartate arninotransferase, alkaline phosphatase, b‐glucuronidase) and plasma bilirubin were in the present study unaffected by the ethanol treatment.

Distribution of lysosomal enzymes between parenchymal and Kupffer cells of rat liver

1. 1. Suspensions of rat liver cells were prepared by perfusing the liver in vitro with solutions containing collagenase and hyaluronidase. About 90% of the liver mass was recovered in the cell suspension. 2. 2. Parenchymal cells were prepared by differential centrifugation or by magnetic removal of iron-loaded Kupffer cells. About 60% of the liver mass was regained in the parenchymal fractions. Specific activity of β glucoronidase (EC 3.2.1.31) was about the same in the parenchymal cells and in liver homogenates. Specific activity of acid deoxyribonuclease (EC 3.1.4.6) in parenchymal cells was about 65% of that in liver homogenates. 3. 3. Kupffer cells were separated from parenchymal cells either by incubating the original cell suspension with pronase, which destroys the parenchymal cells, or by magnetic means. 70 and 40% of the Kupffer cells in the original cell suspension could be recovered by the pronase method and the magnet method, respectively. Iron-loaded cells were contaminated with parenchymal cells. Acid deoxyribonuclease and β-glucuronidase were found to be selectively concentrated in Kupffer cells prepared with pronase. Specific activities of β-glucuronidase and acid deoxyribonuclease were, respectively, 3.6 and 13.6 times higher in pronase-prepared Kupffer cells than in parenchymal cells. Only acid deoxyribonuclease was found to be increased in Kupffer cells isolated by means of a magnet (about twice as high as in parenchymal cells).