The Study Of Phosphodiesterase From The Venom Of Trimeresurus Stejnegeri

The phosphodiesterases (PDEs) are a superfamily of enzymes that have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. PDEs influence a vast array of pharmacological processes, including proinflammatory mediator production and action, ion channel function, muscle contraction, lipogenesis, glycogenolysis and gluconeogenesis. PDEs has been described in a wide variety of snake venoms, and most of them are high molecular mass (>90 kDa) single polypeptide chain proteins. PDEs exhibit broad substrate specificity. They can hydrolyze a variety of biologically important nucleotides such as nicotinamide adenine dinucleotide (NAD), nicotinamide guanine dinucleotide (NGD), ATP and ADP. Though a few PDEs have been isolated from several snake venoms, there is no information on their amino acid or full-length cDNA sequences. As far, the PDE from Trimeresurus stejnegeri venom has not been reported in the literature. Considering the diverse physiological functions of PDEs, we expect to isolation a novel PDE from this venom. Here, we report the purification, structure and enzymatic activity of PDE from the venom of Trimeresurus stejnegeri. The whole thesis is divided into four chapters.The first chapter: It is a brief review about the research progress of PDEs and some research methods for studying protein. After reading this chapter, you can have a whole understanding about the biological function, classification, organizational distributing and crystal structure of PDEs. The structures and enzymatic activities of PDEs in snake venom have also been introduced. The research methods for studying protein, including UV-vis, fluorescence, ion exchange chromatogram, gel filtration chromatogram, polyacrylamide gel electrophoresis, and protein sequencing are also summarized in this chapter.The second chapter: Here we describe for the first time the isolation and characterization of a novel phosphodiesterase from Trimeresurus stejnegeri venom, named TS-PDE, using ion exchange and gel filtration chromatography. TS-PDE is a disulfide-linked dimer with a molecular mass of 100 kDa determined by SDS acrylamide gel electrophoresis. Its isoelectric point was also determined to be 5.1 by capillary isoelectric focusing. The purified TS-PDE is fairly homogeneous as determined by SDS acrylamide gel electrophoresis and capillary isoelectric focusing. We have analyzed the N-terminal amino acids of TS-PDE by Edman degradation. The first four N-terminal amino acids were determined to be Val/Ser, Gln/Leu, Ala/Tyr and Asp/Leu. This result suggests that TS-PDE is a heterodimer and has a unique structure which is different from the known PDEs. TS-PDE is a glycoprotein which contains 2.48 % carbohydrate.The third chapter: Here we investigate the enzymatic activity of TS-PDE and inhibition of ADP-induced platelet aggregation by TS-PDE. The enzymatic activity and kinetic parameters of TS-PDE were assayed with NAD, NGD, ATP, and ADP as substrates by HPLC. The purified TS-PDE exhibits broad phosphodiesterase substrate range with the order of specificity: NGD > ATP > NAD > ADP. In the process of purification of PDE, the ever-present problem is the contamination with other enzymes, such as alkaline phosphatase or 5'-nucleotidase. The 5'-nucleotidase and alkaline phosphatase activities of the purified TS-PDE were determined using AMP and p-nitrophenyl phosphate as substrates, respectively. The purified TS-PDE shows no contamination with either alkaline phosphatase or 5'-nucleotidase activity. The results further indicate that TS-PDE is a phosphodiesterase which has been purified to fair homogeneity. TS-PDE can catalyze the hydrolysis of phosphodiester bonds of DNA, beginning at the 3'-end of nucleotides, liberating 5'-mononucleotides progressively, and may be used as a tool to elucidate the structure and sequence of DNA. In addition, TS-PDE inhibits ADP-induced platelet aggregation and can be used to develop therapeutic agents for the treatment of thrombosis.The fourth chapter: The effects of metal ions and disulfide-bond linkages on TS-PDE activity and structure have been investigated in this chapter. The contents of metal ions in the purified TS-PDE were measured by inductively coupled plasma atomic emission spectrometer (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). Cu²⁺ and Zn²⁺ were detected in the purified TS-PDE. As far as we know, TS-PDE is the one and only enzyme of PDEs which contains Cu²⁺. The PDE activity of the enzyme was almost completely inhibited by EDTA, suggesting that metal ions are essential for the PDE activity of TS-PDE. The metal-free TS-PDE (apo-TS-PDE) can recover its PDE activity in the presence of Cu²⁺ or Zn²⁺, suggesting that both Cu²⁺ and Zn²⁺ are essential for its PDE activity. The native TS-PDE molecule contains seven disulfide-bonds. The all tested small-molecule reductants (DTT,GSH,TCEP and Vc) can almost completely inhibit the PDE activity of TS-PDE. The reduction of disulfide bonds in TS-PDE by DTT, GSH or TCEP is simultaneous with the inhibition of PDE activity, suggesting that disulfide bonds in TS-PDE may play an important role in retaining the enzyme activities. The reduction of Cu²⁺ in TS-PDE by Vc may result in the loss of enzyme activity. The result further indicates that the PDE activity is dependent on metal ions.