Preparation, Enzymological Characters, Characterization Of A Group Of Novel Nucleases From Lumbricus Bimastos

Pharmaceutical and clinical effects of Chinese Medicine earthworm on infectious diseases and our lab's previous research on anti-virus effects of extracts from Lumbricus Bimastus tissues indicated that there are molecular systems comprised of many factors that can inhibit, degrade microbes and single-cell organisms in Lumbricus Bimastus, these molecules cooperate with each other to accomplish the functions of digesting microbes and obtaining nutrition from them. Nucleases are the necessary elements among this molecular system, their functions are not limited to digesting, degrading the genetic materials and killing microbes should owe to them. Little has been studied on the biological significances of nucleases and there is still not an unambiguous conclusion, no study on the relationship between nuclease and immunity and anti-virus effects of innate immunity has been reported. It is noticeable that previous study has proved there are a big quantity of nucleases in the alimentary canals of many creatures, but nucleases were chronically considered to be only related to digesting and absorbing nucleic acids. This viewpoint contradicted with the facts of nucleotides and nucleic acids metabolizing within the bodies. In most animals, the materials needed by nucleotide synthesis are not absorbed from digestive systems, but from amino acids and their metabolizing products through two pathways named as de novo synthesis pathway and salvage synthesis pathway. Study of nutriology do not support this viewpoint also, because nucleotides and their metabolizing products are not essential nutrition for the body. Thus, so many kinds and high-active nucleases in the alimentary canals should have other significant biological functions, such as eliminating the nucleic acids in the food, preventing the invasion of foreign genes, killing viruses and microbes. The restriction enzymes in the prokaryotic organisms support this hypothesis, their gene immune system's main function is maintaining the genetic stability.The relationship between the nucleic acids and immune system was investigated in our lab, the results showed the high activities of nucleases in many types of immune cells, this indicated its close relation with innate immune. In order to verify the types, amount, enzymological characters, structures and biological activities and mechanisms of Lumbricus Bimastus nucleases, explore the universality of its existence in many organisms lived on microbes, the relationship between the nucleases and the mechanisms of degrading internal and foreign senescent cells and microbes, Lumbricus Bimastus was selected as research model, the nucleases in it was purified, their physical, chemical and enzymological characters were studied to explore the activities, amount and distributing of nucleases in many kinds of low forms of life and immune cells.1. Separation and purification of Lumbricus Bimastus nucleases EWDs and EWNsTechniques of enzymology, proteinic research and nuclease analysis were used to investigate the physical, chemical and enzymological characters of the crude extractions that relate to its extraction and purification. Normal pressure and high pressure chromatography were used to study the chromatographical characters and conditions of the preparations with nuclease activity. Neutral and acid conditions, organic solvents, pI precipitation, anion and cation exchange chromatography and PAGE recovery were established as high efficiency and economical pathway for purification. Many nucleases' activities were determined by many analytical techniques. The results showed, in Lumbricus Bimastus, there were 3 kinds of DNase, named respectively EWDl(157kDa),EWD2(69.1kDa), EWD3(62.6kDa), all are electrophoresis purity, 2 kinds of Rnase, named EWN₁ (Earthworm Nuclease 1) and EWN₂ (Earthworm Nuclease 2), all are chromatography purity. The above findings had not been seen before. The study in this section provided adequate proof for the diversity of nucleases in Lumbricus Bimastus, and provided high-purity samples for further enzymological, structural and functional research.2. Enzymatic kinetics characters and the effective factors of Lumbricus Bimastus nucleases EWDs and EWNsIn this section, Sephacryl-200 column chromatography, SDS-PAGE and MADLT-TOF were used to determine the MWs of EWD₁, EWD₂, EWD₃ as 157kDa, 69.1kDa and62.6kDa respectively. Their MWs are apparently larger than DNase I and DNase II. Temperature stabilities, pH stabilities, stimulant and antagonist studies were conducted on EWDs and EWNs, the parameters of these effective factors were different from that of present Dnases. The three EWDs had very similar enzymological characters and stable Physical and chemic characters. Enzymological studies showed Kms of three EWDs were 1.58,3.95 and 1.52 mg /L, this indicated the activities relationship of the three EWDs: EWD₂ > EWD₃> EWD₁. EWNs can degrade RNA, their activities were not dependent on Mg²⁺, Ca²⁺, Mn²⁺. The results above indicated that EWDs and EWNs found in Lumbricus Bimastus had special enzymological characters, their catalytic mechanisms and classification wait to be studied and sorted.3. Action mechanisms of Lumbricus Bimastus nucleases EWDs and EWNsZORBAX-Oligo column HPLC, agarose electrophoresis, UV spectrophotometer analysis were used to investigate the intermediate products and end products of EWDs and EWNs processing circular, linear and single strand DNA. The results showed that EWDs and EWNs can degrade all types DNA, the end products were homogeneous oligo-nucleotides of <18bp without length differences. EWNs can also degrade RNA substrates, EWDs can not. The results indicated that EWDs were endo-deoxynucleotidase (DNases), EWNs were non-specific Nucleases. 4. Biological activities and action mechanisms of Lumbricus Bimastus nucleasesAgarose electrophoresis activity assessing method was used to determined the activities distributing of nucleases in several creatures similar to earthworm and several kinds of immune cells, the results showed the tissue extraction fluids of black worm, daphnia, Tenebrio molitor L contain high-activity Dnase. Nearly all immune cells contain big quantity and high-activity nucleases, for example, high Dnase activity was observed in white blood cells; lymphocytes lysis fluids can degrade circular DNA, it indicated there are Dnase in lymphocytes; CIK cells can degrade plasmid and lamda DNA; macrophages can degrade plasmid,λ-DNA, RNA, this indicated there are Dnase and Rnase in macrophages.Conclusions1. The technique route of Lumbricus Bimastus nucleases extraction and purificationwas established. 3 Dnases were prepared and named EWD1(157kDa), EWD2(69.1kDa), EWD3(62.6kDa), electrophoresis grade purity, 2 kinds of Rnases, named EWN₁(Earthworm Nuclease 1) and EWN₂ (Earthworm Nuclease 2), chromatography grade purity.2. The enzymological kinetics characters and effective factors of Lumbricus Bimastusnucleases were investigated, the results showed the MWs of 3 purified Dnases were 157 kDa,69.1 kD and 62.6 kDa, their substrates is DNA, including double strand DNA, single strand DNA, circular DNA and linear DNA, the optimal pH was between 4.0-5.6, these 3 Dnases are sensitive to temperature, they do not display activity when the temperature rises to 55 degree, Mn²⁺,Ca²⁺ are stimulant for their activities, Mg²⁺ had no significant impact on their activities.3. ZORBAX-Oligo column HPLC and other techniques were used to investigate thesubstrate specificity and degrading characters, the results showed that EWDs and EWNs can degrade all types DNA, the end products were homogeneous oligo-nucleotides of 4-7bp without length differences. EWNs can also degrade RNA substrates, EWDs can not. The results indicated that EWDs were endo-deoxynucleotidase (DNases), EWNs were non-specific Nucleases.4. The tissue extraction fluids of black worm, daphnia, Tenebrio molitor L contain high-activity Dnase. Nearly all immune cells contain big quantity and high-activity nucleases, this indicated they may concerned with the mechanism of the body resist foreign gene's invasions.