Purification and Characterization of Squid Heptopancreatic Nuclease

• Main Authors: Yeh, Hsiao-Ling, 葉筱玲
• Other Authors: Liao, Ta-Hsiu, PhD
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/50859274349305642088

碩士 === 國立臺灣大學 === 生化學研究所 === 89 === Mammalian pancreatic deoxyribonuclease (DNase) is highly specific for DNA as substrate. Shrimp hepatopancreatic nuclease, on the other hand, is a nuclease. Nucleases, are enzymes capable of hydrolyzing both DNA and RNA, in a broad sense, should include DNase, RNase and the sugar-non-specific nuclease. To reveal whether or not the mammalian DNases are evolved from the shrimp nuclease, herein we described the investigation of yet another nuclease from squid hepatopancreas. The hepatopancreatic tissue of squid from Argentina (Illex argentinus) was homogenized and the extract was subjected to ammonium sulfate precipitation followed by chromatography on Macro-Prep High Q, Phenyl Sepharose CL-4B, and Sephadex G-100 columns. The enzyme thus purified was homogenous as evidenced by a single band of Mr= 45 kDa on SDS-PAGE with silver-stain and DNase activity staining showed that the band coincided with DNase activity. Squid nuclease has a specific activity of 4.8x103 units/mg protein, a pH-activity optimum of 7.0 and the best metal ion requirements of 20 mM MgCl2 plus 10 mM CaCl2. Squid nuclease was a very acidic protein (pI= 3.5), but was more stable at higher pH’s. Sugar-affinity chromatography revealed that squid nuclease was not a glycoprotein, a nature similar to shrimp nuclease. Thermal stability experiments showed that squid nuclease had a Tm of about 40 ℃. Like shrimp nuclease, squid nuclease showed at least three forms on SDS-PAGE, depending on the sample treatments, such as in the presence of sodium dodecyl sulfate and β-mercaptoethanol and with or without heating at 95 ºC. The amino acid composition of squid nuclease showed a high content of aspartate and glutamate, in agreement with the acidic nature of the enzyme. Squid nuclease has an intrinsic RNase activity as analyzed by the RNase zymogram method. Thus, like the shrimp enzyme, squid DNase is a nuclease. The N-terminal amino acid residue was blocked and could not be determinated on a protein sequencer. Currently, we are trying to determine the cDNA sequence for squid nuclease with primers designed from the sequences of the peptides derived from formic acid cleavage or trypsin digestion.