Studies on Chloroplast Development and Replication in Euglena: III. A Study of the Site of Synthesis of Alkaline Deoxyribonuclease Induced during Chloroplast Development in Euglena gracilis

During chloroplast development in Euglena, the activity of a specific DNase, Euglena alkaline DNase, increases in a manner similar to that of chlorophyll synthesis, but without the lag customarily associated with the early hours of chlorophyll synthesis. The increase in Euglena alkaline DNase activity is not inhibited by chloramphenicol or by streptomycin, but is inhibited by cycloheximide. Euglena alkaline DNase activity is present in a group of aplastidic substrains which contain carotenoids. These results are interpreted to mean that this chloroplast-related DNase is synthesized in the cytoplasm, and that the genetic information for this enzyme is probably nuclear.     

The characterization of the EBV alkaline deoxyribonuclease cloned and expressed in E. coli.

Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus. We report here the expression of the BGLF5 open reading frame in E. coli and the expression of high levels of a novel alkaline DNase activity in induced cells. This alkaline DNase has been purified to apparent homogeneity as a single protein species. This is the first report of the expression of a herpesvirus coded DNase in a prokaryotic system and of the purification of the EBV DNase to demonstrable purity. It has the biochemical characteristics of a typical herpesvirus alkaline exonuclease showing a high pH optimum, an absolute requirement for Mg2+ for activity and sensitivity to high salt concentrations and polyamines. The enzyme activity was neutralized by sera from patients with nasopharyngeal carcinoma and was reactive with these sera in Western blot analysis. Thus the prokaryotic expression system described here provides an economical and efficient source of the EBV DNase for biochemical and seroepidemiological analysis.     

Thymidine as synchronizing agent. 3. Persistence of cell cycle patterns of phosphatase activities and elevation of nuclease activity during inhibition of DNA synthesis

Synchronous cultures of HeLa cells were obtained by selective detachment of cells in mitosis and fluctuations in enzyme activity were followed during the subsequent cell cycle. The enzymes measured were alkaline and acid phosphatases and a nuclease active on denatured DNA at alkaline pH (alkaline DNase). Each of these enzymes showed a different pattern of activity in the cell cycle, but a temporal relationship to the DNA synthetic phase was apparent in each case. Treatment of the cultures at the beginning of the cell cycle with 15 mM thymidine did not alter the subsequent pattern of fluctuations in activity of alkaline phosphatase or of acid phosphatase, although DNA synthesis was fully inhibited by this treatment. This indicates that the pattern of activity of some enzymes is not linked to DNA replication. On the other hand, the pattern of fluctuations in the activity of alkaline DNase was abolished by thymidine treatment, and elevation of the activity of this enzyme was observed. These results suggest complex and variable relationships between phases of the cell cycle and enzyme activity, and show that inhibition of DNA synthesis is not a suitable procedure for induction of culture synchrony if enzyme activities are to be studied.     

In vivo reactivation of DNases in implanted human prostate tumors after administration of a vitamin C/K(3) combination.

Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K(3) combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-microm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles.     

Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.     

Different deoxyribonucleases in human lymphocytes

The distribution pattern of deoxyribonuclease activities in human lymphocytes has been examined by micro-disc-electrophoresis. Four groups of deoxyribonuclease activities, differing in their electrophoretic mobility, in the nature of their optimal substrate and in their optimal incubation conditions, are characterized. There are two alkaline DNase-activities. One corresponds to DNase I (EC 3.1.4.5), the other having pH optimum of about pH 9.0, prefers denatured DNA as substrate and is not dependent on divalent cations. The fractions with an acid pH optimum can be subdivided into two groups, which differ in their activity towards native DNA, towards denatured DNA, in their activity when succinate is present and in their pH optimum.     

DEOXYRIBONUCLEASES IN DEVELOPING AND ADULT RAT CEREBELLUM: TRANSIENT AND LONG DURATION ACTIONS OF METHYLAZOXYMETHANOL

—Acid and alkaline DNase activities were determined in postnatal and adult rat cerebellum. Normal animals were compared with those injected with 10 mg/kg methylazoxymethanol acetate using varying schedules after birth in order to induce transient damage to proliferating cell populations. In all animals studied, both immature and adult, methylazoxymethanol caused an increase in acidic and decrease in alkaline DNase activity. These effects were not specific to the dividing populations and occurred in nonmitotic cells as well. When injections were given to young adults, these effects appeared irreversible and persisted up to 3 months after birth.     

Alkaline deoxyribonucleases released from Neurospora crassa mycelia: two activities not released by mutants with multiple sensitivities to mutagens.

Three major alkaline deoxyribonuclease (DNase) activities have been identified in sorbose-containing liquid culture medium in which wild-type Neurosporacrassa were grown: DNase A, a Ca++dependent endonuclease of molecular weight 65,000 daltons which has no specificity for single- or double-stranded DNA (ss-DNA or ds-DNA) and no activity with RNA; DNase B, a Mg++-dependent single-strand specific exonuclease of molecular weight 78,000 daltons active with both ss-DNA and RNA; DNase C, a divalent metal ion-dependent endo-exonuclease of molecular weight 65,000 having single-strand specific endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA. Three mutants which were shown previously to have wide spectra of sensitivities to mutagens, and which exhibited reduced release of DNase activity on sorbose-containing agar test plates (the Nuh phenotype), were deficient relative to the wild-type in the release of these major alkaline DNases into the liquid culture medium. The uvs-3 mutant released only small amounts of DNase A and DNase C; nuh-4 did not release detectable DNase C and released only a very low level of DNase B; uvs-6 released only a low level of DNase A. A nuh mutant (nuh-3) which is not mutagen sensitive relative to the wild-type released low levels of DNase B. On the other hand, an ultraviolet light-sensitive mutant (nuc-2) which does not have the Nuh phenotype was normal in the release of these DNases.     

Deoxyribonuclease II from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.     

Deoxyribonucleases from rat brain

Two distinctly different DNases were isolated from rat brain and could be separated easily by ammonium sulphate fractionation. One of the DNases acts optimally at pH 5.0 hydrolysing preferentially native DNA and requiring an optimal Mg²⁺ concentration of about 0.03 m . The other DNase has its optimal pH between 7.4 and 8.9, acts preferentially on heat-denatured DNA and requires a lower Mg²⁺ concentration, the optimum being 1 × 10^?4m . Cerebellum from adult rat brain has a lower acid DNase activity and higher alkaline DNase activity, and therefore has a higher ratio of alkaline DNase to acid DNase than the other areas of brain studied. This unique activity ratio in cerebellum of adult rat brain was not observed in infant rat brain.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase

The pseudorabies virus (PRV) DNase is an alkaline exonuclease and endonuclease, which exhibits an Escherichia coli RecBCD-like catalytic function. The PRV DNA-binding protein (DBP) promotes the renaturation of complementary single strands of DNA, which is an essential function for recombinase. To investigate the functional and physical interactions between PRV DBP and DNase, these proteins were purified to homogeneity. PRV DBP stimulated the DNase activity, especially the exonuclease activity, in a dose-dependent fashion. Acetylation of DBP by acetic anhydride resulted in a loss of DNA-binding ability and a 60% inhibition of the DNase activity, suggesting that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DNase behaved in a processive mode; however, it was converted into a distributive mode in the presence of DBP, implying that PRV DBP stimulated the dissociation of DNase from DNA substrates. The physical interaction between DBP and DNase was further analyzed by enzyme-linked immunosorbent assay, and a significant interaction was observed. Thus, these results suggested that PRV DBP interacted with PRV DNase and regulated the DNase activity in vitro.     

Leishmania mexicana: amastigote hydrolases in unusual lysosomes

Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.     

Functional evaluation of tryptophanyl residues of bovine and porcine pancreatic deoxyribonucleases

Trp-155 in bovine DNase A (EC 3.1.4.5) appeared to be unessential for the enzymatic activity for the following reasons: (1) A unique peptide which suggests the environmental difference of Trp-155 was obtained from porcine pancreatic DNase A. (2) Inactivation of the porcine DNase A by NBS modification was fairly paralleled with a decrease in the CD signal, which is characteristic of the "buried" tryptophan in the hydrophobic region (trp-191 in bovine DNase) but not of tryptophans in the hydrophilic portion. Binding of DNase to the poly I: poly C double helix confirmed the important role of this tryptophan.     

Difference spectra, catalase- and peroxidase activities of isolated parenchymal and non-parenchymal cells from rat liver

The reduced minus oxidized difference spectra from isolated parenchymal and non-parenchymal cells from rat liver indicate that the non-parenchymal cells contain a considerable amount of peroxidase. This interpretation is favoured by the more than 30 times higher specific activity of peroxidase (EC 1.11.1.7) in the non-parenchymal cells as compared to the parenchymal cells. The catalase (EC 1.11.1.6) activity in the non-parenchymal cells is 4 times lower than in the parenchymal cells. These results are consistent with an antimicrobial function of the non-parenchymal cells in liver.     

An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.     

The changes in proviral chromatin that accompany morphological variation in avian sarcoma virus-infected rat cells

The clone All of avian sarcoma virus B77-infected Rat-1 cells comprises both morphologically normal and morphologically transformed derivatives. Transformed subclones, in which virus-specific RNA is readily detectable, contain a provirus that is very sensitive to DNase 1 digestion of chromatin, and show DNase 1 hypersensitive sites at the 5' end of the provirus and in 5' flanking cell DNA. Normal subclones with no detectable virus-specific RNA, whether infected cells that have never been transformed or revertants derived from transformed cells, contain a provirus that is far more resistant to DNase 1 digestion. Moreover this provirus lacks hypersensitive sites at its 5' end, although DNase 1 hypersensitive sites were detected at the 3' end of the provirus in either normal or transformed clones. The pattern of cytosine methylation in the proviral restriction sites of the isoschizomers Msp I and Hpa II differed between transformed and revertant clones; the revertants show additional methylation at some CpG doublets.     

Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates

Porcine spleen DNase II (EC 3.1.22.1), one of the best-characterized DNases II, is subcellularly located in lysosomes because the enzyme is co-sedimented with two of the lysosomal marker enzymes, cathepsin D and acid phosphatase. The physicochemical properties, including the subunit structure, sensitivity to iodoacetate inactivation, native molecular weight and chromatographic behavior, of the DNase II purified from the isolated lysosomes of porcine spleen are indistinguishable from those of the same enzyme purified from the whole porcine spleen homogenate. DNase II can also be extracted from porcine liver with 0.05 M H2SO4 or 0.1 M NaCl and purified from either extract by a series of column chromatographies. The purified liver DNase II from either extract has the same subunit structure (alpha-chain, Mr 35,000 and beta-chain, Mr 10,000) as the purified DNase II of porcine spleen. The two liver extracts as well as the extracts of spleen and gastric mucosa contain DNase II with very similar properties on Sephadex G-100 gel filtration, on acid polyacrylamide gel electrophoresis under non-denaturing conditions, and on isoelectric focusing. The data strongly suggest that, for the same species of animal, the DNase II activities in various tissues are associated with protein molecules of identical structure.     

Alkaline phosphatase activity in cultured urinary bladder cancer cells.

Established cell lines derived from human urinary bladder carcinomas produce heat-stable alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] which resembles the oncofetal enzyme of HeLa S3. Rat bladder cancer cell lines derived from chemically induced tumors produce heat-labile alkaline phosphatase. Corticosteroids and/or hyperosmolality do not influence the enzyme of rodent cells, but induce increased levels of activity in human cells. The increase is most pronounced when human cells multiply in hyperosmolar medium containing prednisolone. Under these conditions a rise of over 100-fold in specific activity is noted. This synergistic effect, not seen with other cultured heteroploid cells, may represent a specific characteristic of cells derived from human bladder tumors.