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1.
Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.  相似文献   

2.
Summary DNase activities that show a non-specific pattern of DNA degradation on agarose gels have been detected in six strains of Streptomyces. All the enzymes were located in the cell wall-cytoplasmic membrane space, and released after protoplast production. Synthesis of these nucleases was dependent on the composition of the growth media. Differentiation on agar plates was affected by the same nutritional conditions that regulate DNAse production. All DNases were deoxyriboendonucleases that required Mg2+ and a low ionic concentration for optimal activity. The molecular masses of native DNases range from 37±2 to 43±4 kDa. The enzymes produced nicks in double-stranded DNA and were unable to digest RNA. The widespread presence, location and characteristics of such nucleases in Streptomyces suggest a common role for these enzymes. Offprint requests to: J. Sánchez  相似文献   

3.
Deoxyribonuclease I in mammalian tissues. Specificity of inhibition by actin   总被引:10,自引:0,他引:10  
Enzymes of the DNase I class, similar to bovine pancreatic DNase I with respect to molecular weight and ionic and pH requirements, were found in various tissues of the rat. Their analysis was facilitated by a method for detection of nucleases in crude extracts after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent renaturation of the enzymes. High levels of DNase I were found in digestive tissues, such as the parotid and submaxillary salivary glands and the lining of the small intestine., Appreciable levels were present in the lymph node, kidney, heart, prostate gland, and seminal vesicle. No activity was found in pancreatic extracts. However, under some conditions, tissues rich in proteases gave poor recovery of DNase I. Fourteen other tissues showed little or no DNase I. Inhibition of various DNase I enzymes by rabbit muscle actin was examined both in gels and in solution. Actin inhibited the bovine parotid DNase I as well as the bovine pancreatic enzyme, but actin did not inhibit any of the DNase I enzymes of the rat. This species specificity of actin inhibition makes it unlikely that the very strong association between monomeric actin and bovine DNase I is of general significance for cellular function.  相似文献   

4.
With the use of a reconstituted poly(ADP-ribosyl)ating enzyme system and three purified nucleases, micrococcal nuclease (MN), bull seminal RNase (BS RNase) and Ca2+, Mg2+-dependent endonuclease (BS DNase), as model acceptor proteins for ADP-ribose, the effect of ionic strength on the modification reaction was examined in detail. When these three nucleases were extensively poly(ADP-ribosyl)ated in this system at a low ionic strength (5 mM Tris), they were all inhibited by about 80% and the chain length of the polymer covalently bound to the nucleases was 13 to 23 ADP-ribose units. The observed inhibition was markedly prevented by increasing the ionic strength in the reaction mixture with a concomitant decrease in the polymer size bound to the nucleases. The NaCl concentrations required for decreasing the extent of the inhibition to half of the maximum were calculated to be 20, 50, and 100 mM for MN, BS RNase, and BS DNase, respectively. These values are similar to the NaCl concentrations required for decreasing the average chain lengths of the polymer to half, suggesting that the length of polymer is closely correlated to the extent of inhibition of these nucleases. DNA-binding affinities of these nucleases, expressed in terms of the NaCl concentrations required for eluting the enzymes from DNA-cellulose, were 140, 280, and 340 mM for MN, BS RNase, and BS DNase, respectively. Considering that maintainance of a ternary complex of poly(ADP-ribose) synthetase, acceptor and DNA may be essential for the modification reaction, the relatively strong salt effect observed in the modification of MN may be explained by its low DNA-binding affinity.  相似文献   

5.
1. It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions. In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied. 2. All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA. The non-digestive conditions were as follows. i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0. At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose. Although S1 and P1 were anionic, they still adsorbed to the column. 3. All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH. The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1. The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength. On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.  相似文献   

6.
We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci.  相似文献   

7.
Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.  相似文献   

8.
Here we review the different apoptotic DNases. From a functional point of view, DNases implicated in apoptosis may be classified into three groups: the Ca2+/Mg2+ endonucleases, the Mg2+-endonucleases, and the cation-independent endonucleases. The first group includes DNase I which has no specificity for the linker region, DNase gamma which has some homology with DNase I, and other DNases which cleave DNA in the linker region. Both DNase I and DNase gamma have been cloned. The other nucleases of this category have dispersed molecular weights. Their sequences are unknown and it is difficult to determine their role(s) in apoptosis. It seems that different pathways are present and that these nucleases may be activated either by caspases or serine proteases. The caspase 3 activated DNase (CAD, CPAN, or DFF40) belongs to the Mg2+-dependent endonucleases. DNase II belongs to the third group of acid endonucleases or cation-independent DNases. We have shown the involvement of DNase II in lens cell differentiation. Recently, the molecular structure of two different enzymes has been elucidated, one of which has a signal peptide and appears to be secreted. The other, called L-DNase II, is an intracellular protein having two enzymatic activities; in its native form, it is an anti-protease, and after posttranslational modification, it becomes a nuclease.  相似文献   

9.
Poly d/[3H]A-r5U/ type of synthetic models of bacteriophage DNAs containing thymine analogues were prepared by DNA polymerase and tested for stability against nucleases /r was a n-alkyl group from methyl to pentyl/. The 5-pentyluracil-containing copolymer was found to be most stable: 50 % degradation with pancreatic DNase, spleen DNase, snake venom phosphodiesterase or micrococcal nuclease required 3–15 times as much time as that of poly d/A-T/.  相似文献   

10.
High levels of nuclease activities were identified in filtrates ofAspergillus cultures after growth in low- but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca2+ and Mn2+ (atpH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A ofNeurospora. However, theAspergillus DNase A did not cross-react with antisera to secretedNeurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression ofAspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutantpalcA. This investigation was supported by Grant A2564 from the Natural Science and Engineering Research Council of Canada.  相似文献   

11.
We determined whether recombinant human growth hormone (rhGH) administration might modulate the enzyme degradative capacity of the muscle lysosomal system and influence muscle growth. Muscle cathepsin D, acid RNase and DNase II activities are determined in the gastrocnemius muscle of rhGH-treated post-weaning female BALB/c mice. Linear regressions were used to analyze the relationships of each enzyme with their respective substrate. GH induced a depletion-recovery response of muscle growth through a mechanism which is similar to catch-up growth. In these conditions, cathepsin D activity decreased with age in all animals (GH: 40%; saline: 79%), showing a substantial developmental decline that could reflect changes in the rate of protein breakdown. However, the degradative capacity of cathepsin D was paradoxically unmodified in rhGH-mice compared with saline mice (according to the enzyme vs. substrate linear regression slope), in spite of the increase in enzyme activity elicited by GH. This suggests that the muscle protein breakdown is not increased by GH-treatment in post-weaning mice. The enhancement of muscle protein deposition as indicated by the augmented muscle cell size (protein:DNA ratio) of rhGH-mice (increased 178% from 25 to 50 days) vs. saline, can be attributed to a higher muscle K(RNA). In contrast, acid RNase and DNase II activities directly participate in muscle RNA and DNA degradation. Both nucleases were inhibited by GH treatment (a decrease of 48% and 63%, respectively, vs. saline at 50 days). The decrease in RNase activity suggests an inverse relation between the rate of protein synthesis (high) and acid RNase activity (low), leading to spare muscle RNA for synthesizing protein during catch-up growth. Also, low DNase II activity could contribute to inhibiting of muscle DNA degradation, facilitating muscle growth. Thus, GH seems to act as a direct modulator of the degradative capacity of skeletal muscle nucleases but not of cathepsin D, influencing DNA and RNA degradation during the depletion-recovery response to GH of gastrocnemius muscle in female post-weaning mice.  相似文献   

12.
The specific activities of acid (pH 5.5) and neutral (pH 7) DNases and RNases were determined in alfalfa (Medicago sativa L.) seedlings grown in the dark in the presence of 3.7 mM paraquat (PQ) or 1 mM roundup (RD). Seedlings were taken at 0, 1, 3, and 5 days. Plant growth parameters (plant height and fresh weight) were dramatically reduced under these conditions of growth comparing to the control (grown in water). The DNase and RNase specific activities of herbicide-treated seedlings were reduced. The reduction of activities ranged by about 50–90 and 15–70% in PQ- and RD-treated seedlings, respectively. In vitro, PQ- and RD-treated nucleic acids [single-stranded DNA (ssDNA), RNA, and plasmid DNA (pl-DNA)] were incubated with acid and neutral nucleases. Both enzymes were isolated and purified from alfalfa seedlings. Electrophoretic analysis on agarose gel of the above incubated mixtures revealed the following: (a) neutral nuclease (pH 7) was capable of hydrolyzing PQ-treated ssDNA while acid nuclease (pH 5.5) was incapable. This could be due to the fact that acid and neutral nucleases displayed different base linkage specificity toward ssDNA; (b) RD formed strong complexes with ssDNA that were unable to be hydrolyzed by both nucleases; (c) in contrast, both enzymes were capable of hydrolyzing PQ- or RD-treated RNA; (d) neutral nuclease was capable of nicking and linearizing both PQ- and RD-treated pl-DNA while acid nuclease had the same activity only toward the PQ-treated pl-DNA; (e) the enzyme activities were not inhibited in the presence of both herbicides. The data suggest that the complexes of PQ or RD with DNA should not be functional substrates of nucleases, and consequently cell processes (e.g., metabolism of nucleic acids, gene expression, replication), in which DNA and nucleases are involved, could be disturbed.  相似文献   

13.
The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.  相似文献   

14.
Streptococcus pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. equisimilis, S. zooepidemicus, Streptococcus group G and L were found to produce deoxyribonucleases (DNases) which were demonstrated using the Toluidine Blue DNA Agar (TDA) described for staphylococcal DNases. The activity of streptococcal DNases increased in the presence of Mg++ and Ca++ ions, the pH optimum was about 7.5 and native DNA was the best enzyme substrate. It is consequently recommended to modify the TDA according to these results for the demonstration of streptococcal DNases. All streptococcal DNases, except the DNase of S. zooepidemicus, were found to be heat-stable. Isoelectric focusing was a convenient technique for separation of streptococcal DNases and for estimation of the pI values of the DNases. S. agalactiae and S. dysgalactiae generally exhibited distinct species specific patterns in the isoelectric focusing experiments. The DNases produced by S. pyogenes were serologically related to the DNases of S. dysgalactiae and Streptococcus group G. A similar relationship was demonstrated between the DNases produced by S. equisimilis and Streptococcus group L.  相似文献   

15.
Apoptosis, which is usually accompanied by DNA degradation, is important not only for the homeostasis of metazoans but also for mammalian development. If DNA is not properly degraded in these processes, it can cause diverse diseases, such as anemia, cataracts, and some autoimmune diseases. A large effort has been made to identify these nucleases that are responsible for these effects. In contrast to Deoxyribonuclease I (DNase I), Deoxyribonuclease II (DNase II) has been less well characterized in these processes. Additionally, enzymes of DNase II family in Trichinella spiralis, which is an intracellular parasitic nematode, are also considered involved in the development of the nematode. We have compiled information from studies on DNase II from various organisms and found some nonclassic features in these enzymes of T. spiralis. Here we have reviewed the characterization and functions of DNase II in these processes and predicted the functions of these enzymes in T. spiralis during host invasion and development.  相似文献   

16.
The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.  相似文献   

17.
The PD-(D/E)XK nuclease domains, initially identified in type II restriction enzymes, serve as models for studying aspects of protein-DNA interactions, mechanisms of phosphodiester hydrolysis, and provide indispensable tools for techniques in genetic engineering and molecular medicine. However, the low degree of amino acid conservation hampers the possibility of identification of PD-(D/E)XK superfamily members based solely on sequence comparisons. In several proteins implicated in DNA recombination and repair the restriction enzyme-like nuclease domain has been found only after the corresponding structures were determined experimentally. Here, we identified highly diverged variants of the PD-(D/E)XK domain in many proteins and open reading frames using iterative database searches and progressive, structure-guided alignment of sequence profiles. We predicted the possible cellular function for many hypothetical proteins based on their relative similarity to characterized nucleases or observed presence of additional domains. We also identified the nuclease domain in genuine recombinases and restriction enzymes, whose homology to other PD-(D/E)XK enzymes has not been demonstrated previously. The first superfamily-wide comparative analysis, not limited to nucleases of known structure, will guide cloning and characterization of novel enzymes and planning new experiments to better understand those already studied.  相似文献   

18.
Summary The alkaline and acid DNase and RNase activity was histochemically investigated in biopsies from the human digestive tube. Activity of these enzymes in the mucosal epithelium in different segments of the digestive tube was compared to the statistical incidence of malignant tumors deriving from this tissue (carcinomas). It was found that the alkaline and acid nucleases activity was very intense in small intestine precisely in this segment where the incidence of carcinomas was low, whereas the low activity of these enzymes in the stomach and large intestine corresponded to the high incidence of carcinomas. This observation confirmed our previously elaborated hypothesis, according to which the low activity of nucleases in normal tissues appeared to be a predisposing factor for malignant transformation.It could be also supposed that the nucleases constitute some kind of double barrier mechanism protecting the genetical stability of the cell against foreign nucleic acid incorporation or production; alkaline nucleases being an extracellular and acid nucleases an intracellular barrier.  相似文献   

19.
Among the myriad of enzymes present in animal venoms, nucleotidases and nucleases are poorly investigated. Herein, we studied such enzymes in 28 crude venoms of animals found in Brazil. Higher levels of ATPase, 5'-nucleotidase, ADPase, phosphodiesterase and DNase activities were observed in snake venoms belonging to Bothrops, Crotalus and Lachesis genera than to Micrurus genus. The venom of Bothrops brazili snake showed the highest nucleotidase and DNase activities, whereas that of Micrurus frontalis snake the highest alkaline phosphatase activity. On the other hand, the venoms of the snake Philodryas olfersii and the spider Loxosceles gaucho were devoid of most nucleotidase and DNase activities. Species that exhibited similar nucleotidase activities by colorimetric assays showed different banding pattern by zymography, suggesting the occurrence of structural differences among them. Hydrolysis of nucleotides showed that 1 mol of ATP is cleaved in 1 mol of pyrophosphate and 1 mol of orthophosphate, whereas 1 mol of ADP is cleaved exclusively in 2 mol of orthophosphates. Pyrophosphate is barely hydrolyzed by snake venoms. Phosphodiesterase activity was better correlated with 5'-nucleotidase, ADPase and ATPase activities than with DNase activity, evidencing that phosphodiesterases are not the main agent of DNA hydrolysis in animal venoms. The omnipresence of nucleotidase and DNase activities in viperid venoms implies a role for them within the repertoire of enzymes involved in immobilization and death of preys.  相似文献   

20.
Ta-Hsiu Liao 《Phytochemistry》1977,16(10):1469-1474
A deoxyribonuclease (DNase), similar to bovine pancreatic DNase, has been isolated from germinating barley. Commerically available malt was used as source of the enzyme. The purification procedure involves (a) ammonium sulfate fractionation (45–65% saturation), (b) CM-cellulose chromatography at pH 4.7 and (c) DEAE-cellulose chromatography at pH 8. DEAE-cellulose separates the enzyme into 4 distinct forms, designed as DNases A, B, C, and D. DNase A and B may be rechromatographed on DEAE-cellulose employing a CaCl2 instead of Tris-HCl gradient. Both forms appear homogeneous on regular and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In addition, both forms have a sp. act. of ca 700 units per A unit at 280 nm, similar to the potency of the pancreatic enzyme. DNase C and D, which are present in relatively small quantities in malt, were not characterized. The MWs of DNases A and B, as estimated by the SDS gel electrophoresis techniques, are near 32 000, slightly larger than that of the pancreatic enzyme. In the presence of either Mn2+ or Mg2+, the pH-activity profile of the barley enzyme is similar to that obtained with the pancreatic enzyme. Like the pancreatic enzyme, barley DNase is protected by Ca2+ from inactivation. The amino acid compositions of the A and B forms are about the same; a comparison of the malt and pancreatic enzymes shows many similarities but major differences in the amounts of glutamic acid, proline and glycine. The hydrolysis products of DNA by malt DNase are indistinguishable from those obtained with pancreatic DNase. Further hydrolysis of these products by snake venom phosphodiesterase shows malt DNase to be a 5′-phosphate producer. Deoxythymidine 3′,5′-di-p-nitrophenyl phosphate, one of the synthetic substrates of pancreatic DNase, is also hydrolysed by malt DNase.  相似文献   

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