Nuclear DNAase of the yeast Candida tropicalis

Using stepwise extraction of chromatin from Candida tropicalis by NaCl (0.1-1.0 M) the protein dissociated by 0.3 and 0.6 M NaCl (fractions 0.3 and 0.6) possessing the DNAase activity were obtained. These DNAases are activated by Mg2+ and cause preferential hydrolysis of heat-denaturated DNA. Fraction 0.3 DNAase has a maximum at neutral values of pH (around 7.0) and causes endonucleolytic hydrolysis of DNA. Fraction 0.6 DNAase causes exonucleolytic hydrolysis of DNA but a maximum at alkaline pH (8.0). The properties of isolated chromatin DNAases of Candida tropicalis differ from those of the known DNAases of the yeast Saccharomyces cerevisiae.     

Study of expression of desoxyribonucleases in mammalian cells by a protein renaturation method in polyacrylamide gel

Analysis of enzymatic activity in polyacrylamide gel is based on highly effective separation of proteins by SDS-electrophoresis with their subsequent renaturation and detection of enzymatic activity. This method was used to study an expression of DNAases in culturing of cells HEK293, NIH 3T3, U937. We have found that in HEK293 cells the nucleases with molecular weights 47 and 45 kDa were expressed. The localization of DNAases in the cell nuclei was shown as well. Induction of apoptosis in HEK293 cells increase the level of p47 DNAase and causes the expression of novel 50 kDa DNAase. We suggested that those discovered DNAases could take part in apoptotic DNA degradation.     

The effect of aflatoxin B 1 , aflatoxin B 2 and sterigmatocystin on nuclear deoxyribonucleases from rat and mouse livers

Certain carcinogens have an effect on the activity of pancreatic deoxyribonuclease I (DNAase I, EC 3.1.4.5). The effect of two potent mycocarcinogens, viz. aflatoxin B₁ and sterigmatocystin, as well as the weak carcinogen, aflatoxin B₂, on the activity of two nuclear DNAases (DNAases I and II) from rat liver was therefore investigated.     

Phospholipase a activity in commercial nucleases. Implications for membrane vesicle isolation

Phospholipase A activity was detected in commercial DNAases I and II and in RNAase preparations. The amount of phospholipase correlates inversely with the degree of nuclease purification. The assessment of the level of phospholipase in commercial nucleases is important in cases where enzymatic properties other than those of DNAases and RNAases are to be investigated and when these preparations are to be used in the isolation of biological membranes.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

Relative biological effectiveness of tritium oxide based on nucleic acid metabolic indices

Relative biological effectiveness (RBE) of tritium oxide, as compared to gamma-rays (137Cs), with regard to LD50/30 is 2,32 +/- 0,69 for rats. The RBE coefficients for tritium oxide are obtained with regard to some indices of nucleic acid metabolism in the thymus and spleen during the dose formation (0-14 days). The RBE of tritium oxide increases with a decrease in radiation dose as determined according to the concentration and content of DNA per organ and activity of thymus DNAases.     

Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method.

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.     

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.     

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.     

Acid and alkaline phosphatases activities in vascular smooth muscle: species differences and subcellular distribution

1. Acid and alkaline phosphatase activities were studied in rat and dog aortic muscle using p-nitrophenyl phosphate (p-NPP) as the substrate. Alkaline phosphatase activity was quite comparable to acid phosphatase activity in rat aortic microsomes as well as further purified plasma membranes, but considerably lower than acid phosphatase activity in dog aortic membranes. 2. Subcellular distribution of acid and alkaline phosphatase activities in these vascular muscles indicated that alkaline phosphatases and a large portion of acid phosphatase activities were primarily associated with plasma membranes and the distribution of acid phosphatase showed little resemblance to that of N-acetyl-beta-glucosaminidase, a lysosomal marker enzyme. 3. The rat aortic plasmalemmal acid and alkaline phosphatase activities responded very differently to magnesium, fluoride, vanadate and EDTA. The alkaline phosphatase was more susceptible to heat inactivation than acid phosphatase. 4. These results suggest that these two phosphatases are likely to be two different enzymes in the smooth muscle plasma membranes. The implication of the present findings is discussed in relation to the alteration of these phosphatases in hypertensive vascular diseases.     

An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization

Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.     

The development of phosphomonesterases in the testes of prepuberal chicks.

1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.     

Effects of nutritional deficiencies during neonatal and post-weaning period on rat intestinal phytase and phosphatase activities

Studies were made of the effects of pre- and post-weaning undernutrition and/or protein deficiency on intestinal phytase and phosphatase activities in albino rats and reversibility of the same by subsequent dietary rehabilitation. Neonatal undernutrition induced by rearing the pups in litters of 16 caused a marked decrease in alkaline phytase activity (as compared to those reared in litters of 8), while acid phytase activity decreased to a lesser extent and acid and alkaline phosphatase activities did not change. When neonatally undernourished rats were subsequently continued on a 4 or a 20% protein diet in restricted amounts (2.5 g/day) for 6 weeks the decreases in the alkaline phytase activity but not in that of acid phytase were further aggravated. Acid and alkaline phosphatases were not influenced by these treatments either. On dietary rehabilitation of these rats for subsequent 6 weeks on a 20% protein diet (ad libitum) acid and alkaline phytase activities of intestine recovered partially. These studies indicate the importance of alkaline phytase activity as a marker of intestinal maturation and is also suggestive of interrelationships between nutrition, intestinal development and its alkaline phytase activity.     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Characterization of deoxyribonuclease activities derived from control and inflammation-associated mouse peritoneal macrophages.

Native DNAase (deoxyribonuclease) activities derived from mouse peritoneal cavity and peripheral blood components were separated, detected, and characterized by electrophoresis into polyacrylamide gels containing DNA, followed by incubation of the gels, and staining of the substrate to reveal only the DNAase activities. Resident peritoneal macrophages contained 12 DNAase-II-like activities that were characteristic of that cell type, whereas lymphocytes and granulocytes each contained five DNAases. Induction of inflammation by peritoneal injection of thioglycollate resulted in changes in macrophage DNAase expression, including: increased total DNAase activity, a decrease in the number of activities from 12 to 11, increased activity of a specific subset of the enzymes, and a change in the apparent size of a specific subset of the enzymes. Electrophoretic and enzymic properties and sensitivity to endo-beta-N-acetylglucosaminidase H indicated that the macrophage activities probably represented charge variants of one or two parent peptide chains.     

Subcellular localization and properties of pyridoxal phosphate phosphatases of human polymorphonuclear leukocytes and their relationship to acid and alkaline phosphatase

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid an alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrigfugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase an was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity, Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase an alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.     

PEG和镍处理对水稻细胞磷酸脂酶活性及再生的影响

以水稻为材料研究了PEG和镍处理对组织培养细胞现分化及其磷酸脂酶活性的影响,结果表明18mg/L或30mg/LPEG,10mg/LNiCl2处理14d均能明显促进细胞再生,但20mg/LiCl2处理降低细胞再生能力,水稻细胞再生率与其酸性磷酸脂酶活性成显著正相关。     

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.     

Alkaline and acid phosphatase activity in murine femoral bone marrow following X-irradiation, or X-irradiation and repopulation with syngenic or allogeneic bone marrow or marrow stroma cells

The activity of alkaline and acid phosphatases in the bone marrow from the femoral cavity was investigated in the following groups of mice: (1) normal (non-irradiated); (2) irradiated with 600 R; (3) irradiated and repopulated with syngeneic bone marrow; (4) irradiated and repopulated with syngeneic marrow stroma; (5) non-irradiated, infused with allogeneic bone marrow (host versus graft reaction, HvG); (6) irradiated and repopulated with allogeneic bone marrow (graft versus host reaction, GvH). In addition, the activity of alkaline and acid phosphatases was examined in bone marrow stromal cultures. In irradiated animals the activity of both enzymes was lower than in non-irradiated ones, repopulation with syngeneic bone marrow restoring it to normal. Repopulation with allogeneic marrow (GvH) resulted in a very deep reduction of alkaline, but not acid, phosphatase. It is postulated that the decrease in bone marrow alkaline phosphatase activity can be a sensitive test for the early GvH reaction, preceding such parameters as splenomegaly. Marrow stroma cultured in vitro also showed very low alkaline phosphatase activity.