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

Abstract

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.

Purification and characterization of deoxyribonuclease from small intestine of camel Camelus dromedarius

“…The similar molecular weights of DNases I were reported for rabbit (35 kDa) [31], porcine pancreas (34.5) [16] and hen pancreas (33 kDa) [18]. On the contrary, DNase II from Euglena gracilis and human small intestine had molecular weights of 45 kD and 28–32 kDa, respectively [8], [23].

Fig. 3SDS-PAGE for purification and molecular weight determination of camel small intestine DNase.

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“…The acidic pH optima at 5.3 and 4.6 were detected for DNase II from Euglena gracilis and porcine pancreas [8]. Human small intestine DNase II had pH optimum at 6.0 [23].

Fig. 4pH optimum of camel small intestine DNase 3.

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“…Takeshita et al [26] reported that the DNase I activity of human, pig, bovine, rabbit, rat and mouse required Mg 2+ and Ca 2+ cations. The inhibition of human small intestine DNase II activity by 20% and 40% in the presence of 10 mM Mg 2+ and Ca 2+ cations was detected [23].…”

“…The Euglena DNase II is stable up to 60 °C for 15 min incubation and its activity was completely lost after 15 min incubation at 80 °C. The low temperature optimum at 30 °C was detected for human small intestine DNase II [23]. The kinetic of the camel small intestine DNase 3 for DNA was determined by Lineweaver-Burk plot (Fig.…”

“…DNase II is found in a wide variety of animal tissues. They are active and present in lysosome and believed to act as a barrier to transfection for DNA or vectors entering the cell by endocytosis [7], [23] and may degrade foreign DNAs and play a role in the replication of DNA [2]. DNase II digested DNA of apoptotic cells after phagocytosis [12].…”