DNase Research Articles

Activities of lysosomal enzymes in rabbit brain with experimental neurofibrillary changes

Rabbits were injected intracerebrally with aluminum salt leading to experimental neurofibrillary change formation as a model of Alzheimer neurofibrillary change. Eleven days after the injection, the brain tissues were excised from the cortex, hippocampus, and cervical region of spinal cord. Five lysosomal enzymes (cathepsin D, β-glucuronidase, acid phosphatase, acid DNase, alkaline DNase) were assayed and compared with the control. Cathepsin D, acid DNase and β-glucuronidase activities increased significantly in all 3 areas of aluminum-injected brain. On the other hand, acid phosphatase and alkaline DNase activities remained at the same level. The results showed the lysosomal enzymes did not change in parallel after aluminum administration, suggesting a role of the increased enzymes in the brain with neurofibrillary changes.

Effect of hydroxyurea on subcellular activities of thymidine kinase in developing and aging rat brain

Hydroxyurea, when injected intraperitoneally at a dose of 1 mg/g body weight, inhibited thymidine kinase activity in developing rat cerebrum (16-day-embryonic) and cerebellum (7-day-postnatal) within a few hours of administration. The inhibition was time-dependent and both cytosolic and mitochondrial thymidine kinases were affected. Under the same conditions, the activities of certain other enzymes concerned with DNA metabolism,viz., DNA polymerase, and acid and alkaline DNases were not inhibited. Further, the addition of hydroxyureain vitro had no effect on the activity of any of the enzymes studied. However, similar treatment given to 2-year-old rat failed to exert any inhibition on either the mitochondrial or soluble thymidine kinase activities in grey and white matter regions of cerebrum and cerebellum. It is inferred that hydroxyurea, apart from its already known effect on ribonucleotide reductase of replicating cells, also affects thymidine kinase.

The influence of aging on lysosomal acid DNase of the rat submandibular gland.

Lysosomal and cytoplasmic fractions were prepared from rat submandibular glands for investigation of the release of lysosomal acid DNase in relation to aging. It was found that the acid DNase activity ratio for cytoplasmic/lysosomal fractions in rats aged 27 months was higher than that in three-month-old rats. The release of acid DNase from the lysosomal fraction by shaking was markedly increased in the fraction from the older animals.

Study of acid DNase in cow snout epidermis by micro slab electrophoresis using acidic and basic gel sheet.

Study of acid DNase in cow snout epidermis by micro slab electrophoresis using acidic and basic gel sheet.

Deoxyribonuclease I sensitivity of DNA replicated in permeable mouse sarcoma cells.

To study chromatin structure at the sites of DNA replicated in permeable cells, deoxyribonuclease I (DNase I) sensitivity of newly replicated DNA in permeable mouse sarcoma cells was compared with that of newly replicated DNA in intact cells. About 35% of the DNA replicated in permeable cells was hypersensitive to DNase I, and the remaining DNA showed the same DNase I sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in intact cells, and was close to that of DNA replicated in the presence of cycloheximide. The sensitivity of DNA pulse-labeled with [3H]deoxythymidine triphosphate by replication in permeable cells was reduced significantly by chasing with cold deoxythymidine triphosphate. The present results suggest that chromatin structure at the sites of DNA replicated in permeable cells is similar to that at the sites of DNA replicated in living cells in the absence of protein synthesis, and that some structural change (possibly toward the maturation) of newly replicated chromatin occurs after the DNA replication in permeable cells.

Activity of acid deoxyribonuclease towards diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate). Stereochemistry of transnucleotidylation reaction.

The (Rp)- and (Sp)-diastereoisomers of thymidyl 3'-(4-nitrophenyl phosphorothioate) (1) were found to act as unusual substrates for acid deoxyribonuclease (DNase II). Instead of the expected thymidine 3'-phosphorothioate, the product resulting from the reaction of (Rp)-1 catalyzed by DNase II was identified as (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), while that from (Sp)-1 has been recognized as a 10:1 mixture of (Sp, Rp)-thymidyl (3'-5')thymidyl phosphorothioate 5'-(4-nitrophenyl phosphorothioate) and (Rp, Sp)-thymidyl (3'-5')-thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate), respectively. Both types of transnucleotidylations were found to occur with retention of configuration at phosphorus. Stereochemical results may be interpreted in terms of two step mechanisms involving the formation of the intermediate, covalent substrate enzyme complexes.

Some properties of acid DNase isolated from actinia Radianthus macrodactylus tentacles

1. 1. Acid and alkaline DNases have been found in actinia Radianthus macrodactylus tentacles cell lysate. 2. 2. The acid DNase isolated from cell lysate has a pH optimum at 5.0. The enzyme was inhibited by 20 mM Mg 2+, 50 mM Ca 2+, but was active even at 800 mM NaCl. 3. 3. The enzyme preferentially hydrolysed native DNA with forming the oligonucleotides carrying 3′ terminal phosphate.

The subunit structure and active site sequence of porcine spleen deoxyribonuclease.

An acid DNase (DNase II) from porcine spleen was purified by sequential chromatography over carboxymethyl-cellulose, blue dextran-Sepharose, hydroxylapatite, and sulfoxyethyl-cellulose. The purified enzyme shows two polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 35,000 (alpha chain) and 10,000 (beta chain). The sum of the two molecular weights is that of the native enzyme (45,000). Thus, the DNase II molecule is an alpha,beta dimer. The two polypeptides are not joined by disulfide bonds, but can be cross-linked chemically with dimethyl suberimidate. They are dissociable in 8 M urea, after which they can be isolated by gel filtration on Sephadex G-100, eluting with 1 M acetic acid. Once dissociated, the two polypeptides cannot be reassociated to regenerate DNase II activity. The sum of the amino acid compositions of the two polypeptides is that of the native enzyme, and both contain carbohydrate. The beta chain is devoid of histidine, half-cystine, valine, and methionine. The NH2-terminal amino acid of the alpha chain is leucine, while that of the beta chain cannot be identified by either dansylation or Edman degradation. Alkylation of an essential histidine residue of DNase II occurs on incubation of the enzyme with [2-14C] ICH2COOH (Oshima, R. G., and Price, P. A. (1973) J. Biol. Chem. 248, 7522-7526). Radioactivity is found only in the alpha chain. After hydrolysis of the alpha chain with trypsin, chymotrypsin, and thermolysin, radioactive peptides were isolated by gel filtration on Sephadex G-25 and reversed-phase high performance liquid chromatography. Sequence analyses of the radioactive peptides show alkylation of 1 of 9 histidines in the entire amino acid sequence of DNase II. The sequence around this histidine, determined by manual microsequencing and by the release of amino acids with carboxypeptidases A and B, is Ala-Thr-Glu-Asp-His-Ser-Lys-Trp.

Effect on lysosomes of invertase endocytosed by rat-liver.

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.

Cerebellar deoxyribonucleases and DNA in the androgenized female rat.

Postnatal developmental patterns of activity for acid and alkaline deoxyribonuclease (DNase) were determined in the cerebellum of the androgenized female rat. Littermates were injected s.c. with either 1.25 mg testosterone propionate (TP) or vehicle 48 hours after birth. The enzymes and DNA content were measured in the cerebellum at 3, 6, 10, 15, 18, 22, 35, and 60 days of age. Neonatally administered TP affects neither the activities nor the developmental patterns of the DNases through age 60 days. However, the DNA content was significantly lower in the cerebellum of the TP-treated rats at ages 10 and 15 days. These data demonstrate that the TP-induced alterations in cerebellar DNA content are not mediated by acid or alkaline DNase.

Biochemical diagnosis of reduced salivary gland function

The volume of the secretion of saliva is decreased in elderly people. The volume of salivary secretion varies directly with acid DNase activity. Neutral DNase activity, however, shows no significant variation.

Acid hydrolases during Artemia development: A role in yolk degradation

1. 1. A broad group of acid hydrolytic activities have been determined during Artemia development including cathepsin B acid ribonuclease, acid deoxyribonuclease, acid phosphatase, acid phosphodiesterase, β-glucosidase, β- N-acetylgalactosaminidase and acid lipase. These enzymes present maximum activity in nauplii, when yolk degradation is maximum. 2. 2. Artemia cathepsin B proteinase is able to degrade lipovitellin in vitro with a pattern similar to that found in vivo. These results suggest the involvement of acid hydrolases in the degradation of yolk. 3. 3. In cryptobiotic embryos, acid hydrolases were found associated with structures of buoyant density 1.18. The structures appeared at the electron microscope as 0.4 μm dia. vesicles when revealed by acid phosphatase cytochemistry.

Relationships between forms of deoxyribonuclease I from several mammals

Relationships between forms of deoxyribonuclease I from several mammals

Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase.

Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [3H]thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

Effect of hyperphenylalaninemia induced during suckling on brain DNA metabolism in rat pups.

We studied DNA metabolism (synthesis and degradation) in brain to investigate the effect of hyperphenylalaninemia induced in rats by treatment with PCPA or alpha MPA plus PHE during suckling (4th-20th days of postnatal age) on cell proliferation and naturally occurring cell death. The incorporation of 14C in DNA as percent of total radioactivity in the tissue, 30 min after administration of [14C]thymidine served as a measure of DNA synthesis in vivo, and the amount of radioactivity recovered in DNA as percent of total 14C in the tissues of 21 day old rats, injected with [14C]thymidine on 2nd day after birth, indicated the turnover (degradation) of DNA. The results showed that the DNA content of cerebellum as well as cerebrum was reduced by treatment with PCPA plus PHE, while treatment with alpha MPA plus PHE had no effect on DNA content in cerebellum but reduced the levels in cerebrum. Treatment with PCPA or alpha MPA plus PHE reduced the synthesis of DNA in cerebrum of 11 day old rats but not in 21 day old rats, and the treatments did not affect DNA synthesis in cerebellum of either 11 or 21 day old rats. The turnover (degradation) of DNA was increased in both cerebellum and cerebrum from rats treated with PCPA plus PHE but alpha MPA plus PHE treatment did not alter the DNA turnover either in cerebellum or in cerebrum. The activity of acid DNase was reduced in both cerebellum and cerebrum from 11 as well as 21 day old rats treated with PCPA plus PHE, but the enzyme activity was not altered in the tissues from rats of both ages treated with alpha MPA plus PHE. The data thus indicate that in rats treated with PCPA plus PHE the reduction in cerebral DNA levels occurs due to reduced synthesis and/or increased turnover (degradation) of DNA but that the reduction in cerebellar DNA may occur only as a result of increased turnover (degradation), and that in rats treated with alpha MPA plus PHE the reduction in cerebral DNA must occur due to reduced synthesis. This suggests that treatment of rats with PCPA plus PHE during suckling inhibits cell proliferation and/or increases naturally occurring cell death in both cerebellum and cerebrum while treatment with alpha MPA plus PHE inhibits only cell proliferation and in cerebrum alone.

Age-dependent conformational changes in acid deoxyribonuclease of chick brain

Acid deoxyribonuclease (EC 3.1.4.6) (DNase) from young (16 days of incubation) and old (1.5 years) chick cerebral hemispheres was purified to apparent homogeneity. Throughout the purification schedule, the behavior of “young” and “old” enzymes was similar. However, the specific activity of the purified enzyme from old brain was only one-tenth that of young enzyme. Polyacrylamide gel electrophoresis of the purified acid DNase gave a single band. Antisera against both “young” and “old” enzyme were raised and double immunodiffusion experiments revealed cross-reaction of young antigen with old antiserum and vice versa, although precipitin bands with young antigen against young antiserum and old antigen against old antiserum were more sharp. Both young and old acid DNase preparations showed an apparent molecular weight of 62 000 and many other properties like heat stability, effect of various exogenous compounds like Hg 2+, Zn 2+, Mg 2+, etc., were also similar. The old enzyme showed slightly higher K m and decreased V max compared with the young enzyme. Dansylation of N-terminal amino acids and their analysis following tryptic digestion of both “young” and “old” acid DNase revealed a similar pattern. Immunotitration experiments showed that the old enzyme requires more antiserum prepared against “young” enzyme to achieve 50% inactivation, thus pointing out the presence of completely or partially inactive molecules in “old” acid DNase preparation. Circular dichroism spectra of the enzyme preparations indicated that the “old” acid DNase molecules are more rigid and have more δ-helical structure, compared with the “young” enzyme. From these data, it is suggested that the reduction in the specific activity of old acid DNase may be, apart from other possibilities, due to conformational changes in the enzyme molecules.

Lysosomal acid deoxyribonuclease from vervet monkey livers—I: Purification and physico-chemical characterization

1. 1. Acid DNase from monkey liver lysosomes was purified to homogeneity by salt extraction of lysosomal membranes at pH 3.8; (NH 4) 2SO 4 fractionation; low salt precipitation; SP-C 50 and G-150 Sephadex chromatography; and polyacrylamide gel electrophoresis. 2. 2. The pH for optimum activity was dual in character with a labile optimum at pH 3.8 and a less active but stable one at pH 4.2 3. 3. The estimated molecular weight was 40 K and the p l was 4.4. 4. 4. Inorganic ions such as Ca 2+, Mg 2+, Mn 2+ and SO 4 2− were more than 80% inhibitory at 10-mM levels. Fe 3+ ions were 80% inhibitory at 0.1-mM levels. 15. Nad at 100 mM is essential for activity but becomes 100% inhibitory above 200 mM.

慢性関節リウマチ関節液中の酸性ＤＮＡ分解酵素について （第一報）

An acid deoxyribonuclease activity was detected in synovial fluids of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) by sensitive method using 32P-labeled Escherichia coli DNA as substrate for enzyme assay. Nineteen patients, 20 joints with RA and 7 patients, 10 joints with OA were investigated. The enzyme activity was dected in only 3 of 9 cases with RA classified in stage I and II of Steinbrcker's stage classification. The enzyme activity was detected in 4 of 5 cases with stage III and all of 6 cases with stage IV, most cases of which showed high enzyme activity. The higher enzyme activity in synovial fluids was observed in the cases with more advanced X-ray stage.

Lysosomal acid deoxyribonuclease from vervet monkey livers—II: Enzymic properties

1. 1. Primate liver lysosomal acid DNase is an endonucleolytic enzyme. 2. 2. The enzyme has both 3'- and 5'-nucleotidohydrolase activities. 3. 3. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long. 4. 4. The Arrhenius plot shows a discontinuity with a transition temperature at 47°C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature. 5. 5. The activation enthalpy is 104kJ/mol and the entropy −0.498 kJ/mol/K. 6. 6. The enzyme is subject to substrate inhibition and the K m value is 159 × 10 −3mM DNA-P.

Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon.

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.