ABSTRACT
We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.
Subject(s)
RNA, Ribosomal/chemistry , Ribonucleotides/isolation & purification , Ribosomes/chemistry , Animals , Anion Exchange Resins/metabolism , Carcinoma, Ehrlich Tumor/chemistry , Chromatography, Ion Exchange , Hydrolysis , Kidney/chemistry , Liver/chemistry , Mice , RNA, Ribosomal/analysis , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 28S/chemistry , Rats , Resins, Synthetic , Ribonucleotides/analysisABSTRACT
CL-Sepharose 4B column chromatography has been used for the separation of four major classes of mammalian nucleic acids in a single chromatographic run. Gel filtration at 2.5 M NaCl separated DNAs (containing RNA hybrids) from tRNAs. The 18 S RNA (containing 3-5 wt% of small 5 S RNA and RNA degradative products) was eluted at 0.7 M NaCl, and 28 S RNA (containing hnRNAs) was eluted at 0.1 M NaCl. Poly(A)+ mRNAs were detected in both 18 and 28 S RNA fractions. The present procedure is suitable for both analytical and preparative work and may serve as an initial step for the further isolation of ultrapure nucleic acid preparations.
Subject(s)
Chromatography, Agarose/methods , Chromatography, Gel/methods , DNA/isolation & purification , RNA/isolation & purification , Animals , Carcinoma, Ehrlich Tumor/analysis , DNA, Neoplasm/analysis , Kidney/analysis , Liver/analysis , Mice , RNA, Neoplasm/analysis , RNA, Ribosomal/isolation & purification , RNA, Transfer/isolation & purification , Rats , Sepharose/analogs & derivatives , Spectrophotometry, UltravioletABSTRACT
The examination was made on the effects of adenosine and inosine on glycolysis in ACD stored human red cells. Total glycolytic capacity was checked by the incorporation of inorganic phosphate into the acid soluble nucleoside phosphates of erythrocytes. Adenosine and inosine increase the glycolysis in ACD stored human red cells. This stimulation depends on the nucleoside concentration used up to the saturation level (2.0 muM of adenosine or inosine/ml). By the saturation level it was demonstrated that adenosine gives 30%higher stimulation than inosine. It may be concluded that by the same conditions adenosine may serve as a better corrigens for ACD stored human blood.
