New sequence-specific human ribonuclease: purification and properties.
A new sequence-specific RNase was isolated from human colon carcinoma T84 cells. The enzyme was purified to electrophoretical homogeneity by pH precipitation, HiTrapSP and Superdex 200 FPLC. The molecular weight of the new enzyme, which we have named RNase T84, is 19 kDa. RNase T84 is an endonuclease which generates 5'-phosphate-terminated products. The new RNase selectively cleaved the phosphodiester bonds at AU or GU steps at the 3' side of A or G and the 5' side of U. 5'AU3' or 5'GU3' is the minimal sequence required for T84 RNase activity, but the rate of cleavage depends on the sequence and/or structure context. Synthetic ribohomopolymers such as poly(A), poly(G), poly(U) and poly(C) were very poorly hydrolysed by T84 enzyme. In contrast, poly(I) and heteroribopolymers poly(A,U) and poly(A,G,U) were good substrates for the new RNase. The activity towards poly(I) was stronger in two colon carcinoma cell lines than in three other epithelial cell lines. Our results show that RNase T84 is a new sequence-specific enzyme whose gene is abundantly expressed in human colon carcinoma cell lines.
Institut National de la Santé et de la Recherche Médicale Unité 467, Centre Hospitalier Universitaire Necker,75015 Paris, France. greg@nencki.gov.pl
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Przewlocki
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Lipecka
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Edelman
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Przykorska
D000230:Adenocarcinoma / Q000201:enzymology*; D001483:Base Sequence; D014408:Biomarkers, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology*; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011063:Poly A-U / Q000378:metabolism; D011069:Poly I / Q000378:metabolism; D011131:Polyribonucleotides / Q000378:metabolism; D012349:RNA, Transfer, Asp / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D011131:Polyribonucleotides; D012349:RNA, Transfer, Asp; D012360:RNA, Transfer, Phe; C114717:poly r(A-G-U); D011063:Poly A-U; D011069:Poly I; D004722:Endoribonucleases; C114716:ribonuclease T84