Mechanism of action of deoxyribonuclease II from human lymphoblasts

Harosh, Itzik; Binninger, David; Harris, Paul V.; Mezzina, Mauro; Boyd, James B.

doi:10.1111/j.1432-1033.1991.tb16397.x

Cited by 34 publications

(16 citation statements)

References 16 publications

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“…The enzyme was an endonuclease which had an optimal pH and temperature of 6.0 and 30°C, respectively and had one polypeptide chain (monomer) and a molecular mass of 28-32kDa. Most molecular masses of DNase II varied from 26 to 45 kDa (32,34) and compared with other human purified DNases II: DNases from human urine [35], human gastric mucosa and cervix [36], human lymphoblasts [32], and from an enzyme with a DNase I-like structure exhibiting acidic DNase II-like activity from human [37] had 32 kDa, 38 kDa, 45 kDa and 35 kDa respectively. The structures of DNase II family had not been elucidated but several researchers suggested that most of them consisted of a single polypeptide [35,36] similar to the purified DNase from the lysosomes of small intestinal, but the DNase from the human liver consisted of three non-identical subunits [38].…”

Section: Discussionmentioning

confidence: 99%

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A Novel Method For Purifying a DNase From Lysosomal Fraction From Human Small Intestine

Shikara¹,

Al-jaff²

2012

Turk J Bioch

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Aims:The purification of an acid DNase related DNase II family from lysosomes of human small intestine (jejunum). Materials and Methods: Two different methods: a column chromatography series, including phosphocellulose, CM-cellulose and Sephadex G-200 and a novel procedure that use immunoabsorbent technique were used in the purification. Results:The purified DNase is an endonuclease that consisted of one polypeptide chain (monomer) with a molecular mass of 28-32kDa with an optimal pH and temperature of 6.0 and 30°C, respectively. The enzyme prefers native DNA on denatured DNA Discussion: The catalytic properties of the purified enzyme are essentially the same as those of DNase II family such as independency of divalent ions which inhibited its activity at 10mM. The enzyme acts on dsDNA and ssDNA and generates 3'-phosphate and 5'-OH termini which was a characteristic of DNase II. The functions of the intestinal purified DNase from lysosomes were unclear, but lysosomes contained a set of enzymes required for degradation of food, and the enzyme may be necessary for degradation of nucleic acids within food, but small amounts of the enzyme were found in the nuclei and cytoplasm were detected. Several researchers suggested that DNase II family may be active in apoptosis and played a role as a barrier to transfection. The purified was designated SIDNase. Key words: DNase II, small intestine, immunoabsorption, lysosomes, DLAD Conflict of interests: There is no conflict in interest. ÖZETAmaç: İnsan ince bağırsağından (jejunum) asit DNaz II ailesi ile ilintili DNaz izolasyonu. Materyal ve Metod: İzolasyonlar için iki farklı metod uygulanmıştır: Fosfoselüloz CMselüloz ve Sephadex G-200 den oluşan bir kolon serisi ve immünabsorbsiyon tekniği kullanılarak geliştirilen yeni bir prosedür. Sonuçlar: İzole edilen DNaz tek bir polipeptit zincirinden (monomer) oluşan bir endonükleaz olup, moleküler ağırlığı 28-32 kDa, fonksiyonu için optimal ısısı 30°C, pH ise 6.0 dır. Bu enzim natif DNA'yı denatüre DNA'ya tercih etmektedir. Tartışma: İzole edilen enzimin katalitik özellikleri DNaz II ailesine ait enzimler ile temelde aynıdır. Mesela iki değerlikli iyonlardan bağımsız aktivite göstermesi, aktivitesinin 10mM da inhibe olması gibi. Bu enzim dsDNA ve ssDNA üzerinde DNaz II gibi 3'-fosfat ve 5'-OH terminali oluşturur. Bağırsaklardan izole edilen lizozomal DNaz'ın fonksiyonları tam olarak nitelenememiştir ancak lizozomler gıda parçalanması için bir set enzim içermektedir ve gıdada bulunan nükleik asitlerin parçalanması için gerekli olabilirler. Az miktarda enzim çekirdek ve sitoplazmada bulunmuştur. Bazı araştırmacılar DNAz II ailesinin apoptozda aktif olabileceğini ve görevinin transfeksiyonda bariyer oluşturmak olabileceğini öne sürmüşlerdir. İzolat SIDNaz olarak adlandırılmsiyonıştır.

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Section: Discussionmentioning

confidence: 99%

“…DNase I digested products are cleaved by phosphodiesterase I, but not by phosphodiesterase II. This was a clear indication that the purified DNase produced oligonucleotides with a phosphate group at the 3'-termini and a hydroxy group at the 5'-termini, which was a characteristic of DNase II [31,32,33].…”

Section: Terminal Products Produced By the Purified Dnasementioning

confidence: 99%

A Novel Method For Purifying a DNase From Lysosomal Fraction From Human Small Intestine

Shikara¹,

Al-jaff²

2012

Turk J Bioch

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“…A digitized image analyzer (kindly provided by Dr. B. Palcic, BC Cancer Research Centre, Vancouver, BC) was then used to measure the intensity of the signals corresponding to AP-1 DNAbinding activity. Zymography assays were done according to 34 with some modifications. Ten per cent Laemmli SDS ± acrylamide gels were co-polymerized with calf thymus DNA (0.2 mg/ml) and stored overnight at 48C.…”

Section: Methodsmentioning

confidence: 99%

Activation of a low pH-dependent nuclease by apoptotic agents

Famulski

Macdonald²,

Paterson

et al. 1999

Cell Death Differ

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Intracellular acidification caused by agents such as UV(C), etoposide or ceramide accompanies the progression of apoptosis. It is suggested that cellular acidosis may set favorable conditions for a dormant, low pH-dependent (acidic) nuclease, which could be involved in intranucleosomal genome degradation, a hallmark of programmed cell death. Here we show that exposure of HL-60 cells to acidotic/ apoptotic agents results in the several-fold activation of a novel low pH-dependent (acidic) nuclease activity, as revealed by zymography. Its activity, which resides in nuclei, is associated with four polypeptides with apparent M r of 56, 48, 45 and 40 kDa. Treatment of HeLa cells with UV(C) or ceramide causes also the up-regulation of an acidic nuclease activity which is represented by 70 and 62 kDa polypeptides. These observations suggest that acidic nuclease activation can be induced by the same apoptotic agents in different cell types. In HL-60 cells, acidic nuclease up-regulation triggered by acidotic agents follows the induction of AP-1 transcription factor active complexes and accompanies the progression of apoptosis. Inhibition of AP-1 factor activity caused by either anti-caspase/anti-acidotic agent Zn 2+ or curcumin, an inhibitor of AP-1 binding to DNA and c-jun synthesis, protects cells from genome destruction. Acidic nuclease activation, however, is only partially inhibited by these factors. We propose that (i) the up-regulation of an acidic nuclease activity is governed by a regulatory pathway different from that responsible for AP-1 factor induction, caspases activation and intracellular acidification, and (ii) activation of an acidic nuclease does not cause any deleterious effects when AP-1 transcription factor induction, caspases activation and intracellular acidification are down-regulated. Thus, the acidic nuclease up-regulation alone is not a sufficient prerequisite for apoptosis.

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“…Hitherto, DNase II has been only partially purified from gastric mucosa, uterine cervix (18), urine (7,19), and lymphoblasts (20). We previously developed a sensitive and specific method for the quantitative detection of DNase II activity in human tissues and body fluids (7) and were consequently able to discover that human urinary and leukocyte DNase II shows genetic polymorphism with respect to its activity levels (21).…”

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confidence: 99%