Polynucleotide:Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA.
Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose((R)). Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG.
Barbieri
L
L
Department of Experimental Pathology, University of Bologna, Via San Giacomo 14, I-40126 Bologna, Italy. barbieri@alma.unibo.it
Valbonesi
P
P
Righi
F
F
Zuccheri
G
G
Monti
F
F
Gorini
P
P
Samorí
B
B
Stirpe
F
F
D002850:Chromatography, Gel / Q000379:methods; D004396:Coloring Agents; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases; D004278:DNA, Superhelical / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D000922:Immunotoxins; D018625:Microscopy, Atomic Force; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D054788:Ribosome Inactivating Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; D000076985:Saporins; D012639:Seeds / Q000737:chemistry; D012685:Sepharose
D004396:Coloring Agents; D004278:DNA, Superhelical; D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; C025674:GEL protein, Gelonium multiflorum; D004247:DNA; D012685:Sepharose; D003851:Deoxyribonucleases; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; D000076985:Saporins