Purification and Characterization of Tilapia (Oreochromis mossambicus) Deoxyribonuclease I

Abstract

DNase I of tilapia (Oreochromis rnossambicus) was purified to homogeneity. Tilapia DNase I is most active at pH 8.5 with Mg" as activator. The CaZ+/Mg2+ pair has a synergistic effect on activation. The enzyme is readily inactivated by heating above 55 "C, but is not inactivated by trypsin or 2-mercaptoethanol under alkaline conditions, with or without CaCI,. Its isoelectric point is 6.0. The 258-amino-acid sequence of tilapia DNase I was derived from overlapping sequences of tryptic, chymotryptic and CNBr peptides. The purified enzyme has two variants differing by a single Lys+Arg mutation at position 125. The polypeptide chain has one disulfide bridge and one carbohydrate side chain. By mass spectrometry, the purified enzyme shows many molecular mass forms differing by Lys/Arg substitution and sugar-chain length. [5]. The enzymatic properties of DNase I in these three species are very similar, and their primary structures are highly conserved [6]. It has been reported 171 that although the enzymatic properties of shrimp DNase I are very similar to those of mammalian DNase I, the protein structure is very different. Due to its high content of disulfide bonds and a highly cross-linked structure, shrimp DNase I is relatively stable to heat or SDS treatment [8]. A DNase-I-like enzyme, which is inhibited by muscle and liver actin, has been isolated from carp liver [9]. We investigated fish DWase 1 because fish are vertebrates but have a fused hepatopancreas similarly to shrimp. RNA was isolated from a piece of fresh small intestine of the fish with TRIzol Reagent (Life Technologies). Iodoacetic acid and P-mercaptoethanol were obtained from Wako. DEAE-cellulose (DE-52) was from Whatman. Hydroxyapatite (DNA grade) was from Bio-Rad. Trypsin and phenyl-Sepharose 4B were from Sigma. Sephadex G-I 00, ampholytes and concanavalin-A-Sepharose were from Pharmacia. Purified bovine DNase I was obtained via the method described previously [lo]. MATERIALS AND METHODS MaterialsDNase I assay. DNase I activity was determined by the hyperchromicity method [3, 101. 1 U enzyme activity causes an increase in absorbance of 1 at 260 nm in a 1-cm cuvette, per min per ml of assay mixture at 25°C.Purification of tilapia DNase 1. Approximately 450 g of tilapia hepatopancreas was minced and homogenized with a homogenizer (Heidolph DIAX 600) in 400ml 20mM TrislHCl, pH 8.0. Cell debris were removed by centrifugation in a Beckman JA14 rotor at 15 000 rpm for 1 h. The supernatant was filtered through two layers of cheese cloth, and the clear solution, termed crude extract, was subjected to (NH,),SO, fractionation between 35 % and 65 70 saturation. The precipitate obtained was dissolved in SO ml 20 mM Tris/HCl, pH 8.0, and dialyzed against the same buffer for 12 h. The dialyzed solution was passed through a DEAE-cellulose column (5 cmX15 cm), and the column was washed with 900ml of the same buffer. The absorbed proteins were eluted with a linear gradient of NaCl from 0 to 0.2 M at a flow rate of 60 ml/h. DNase I was eluted in approximately 0.05 M ...

Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

“…The brDNase(C101A) mutant, losing the “nonessential” disulfide, was inactivated at a transition temperature of 60°C. Fish DNase, also lacking the “nonessential” disulfide, exhibited a similar transition temperature and was relatively thermal‐labile (Hsiao et al 1997). The transition temperature of 73°C for the mutant with an extra disulfide [brDNase(F192C/A217C)] was similar to the transition temperature for chicken DNase, which, having the corresponding third disulfide, was thermally more stable than bpDNase (Hu et al 2003).…”

“…During molecular evolution of proteins, a change in the number of disulfides in DNase occurs. Thus, fish DNase (Hsiao et al 1997) contains only the “essential” disulfide, and chicken DNase (Hu et al 2003), with the same two disulfides as in bpDNase, has one extra disulfide (C192–C217). Because enzymatic and physical properties of DNase from these three species were not quite the same, it prompted us to investigate, by site‐directed mutagenesis in bpDNase, the possible biological functions of the two disulfides and an engineered disulfide corresponding to the third disulfide present in chicken DNase.…”

Structure and Function of Bovine Pancreatic Deoxyribonuclease I

“…3B), changes in the number of disulfides in DNase occur. Thus, fish DNase [64] contains only the "essential" disulfide and chicken DNase [61], with the same two disulfides as in bpDNase, has one extra disulfide (Cys192-Cys217). Since enzymatic and physical properties of DNase from these three species were not quite the same, it prompted us to investigate the possible biological functions of the two disulfides and the disulfide corresponding to the third disulfide present in chicken DNase [66].…”

“…Nevertheless, the two variant proteins retained the characteristics of the Ca 2+ -induced conformational changes and the Ca 2+ -protection against the β-mercaptoethanol disruption of the essential disulfide bond, suggest- (B) Sequence homology within the motifs of Sites I and II of DNases from various species. Sequences were taken from bovine, ovine, porcine [55], human [56], canine [57], rabbit [58], rat [59], mouse [60], chicken [61], snake [62], frog [63], and fish [64] DNases.…”

Molecular evolution of shark and other vertebrate DNases I

“…Thus, shark DNase I cDNAs appear to be characterized by a shorter 3′‐UTR (average of 96 bp) than those cloned from most other vertebrates (200 ± 89 bp), including the human [29], rabbit [13], mouse [14], rat [30], cow [31], hen [17], pig [15] and snake [18] enzymes. In this respect, shark DNases I resemble the amphibian (89 ± 22 bp) [19] and Osteichtye (112 ± 20 bp) [20,21] enzymes. It could therefore be postulated that the 3′‐UTR of vertebrate DNase I cDNA lengthened about twofold during the evolutionary stage between amphibians and reptiles.…”