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PubMed1983

Purification of isoacceptor transfer RNA by affinity coupling to aldehyde-containing matrix.

A method has been developed for the affinity coupling of aminoacyl-tRNA to an aldehyde-containing polymer by means of reduction with sodium cyanoborohydride. Sephadex dialdehyde obtained from periodate oxidation of Sephadex G-50, and Enzacryl polyaldehyde obtained from hydrolysis of Enzacryl polyacetal, were found to provide the requisite polymeric matrix. This coupling reaction permitted the rapid purification of isoacceptor tRNAs of Escherichia coli for the alpha-amino acids glycine, valine, and arginine, as well as the alpha-imino acid proline.

Amino Acids↗
PubMed1983

A ribonuclease assay: separation of product from undegraded aminoacyl-transfer RNA substrate.

A new rapid microassay for RNase is based on the extraction of the products of the degradation of aminoacyl-tRNA into ethyl acetate. The substrate, for example, f[3H]Met-tRNA, is not soluble in ethyl acetate but the product f[3H]Met-nucleotide is extracted with high efficiency over a wide range of pH in phosphate buffer of high ionic strength. The limit digest of f[3H]Met-tRNA by RNase T1 in which the minimum-sized product should be f[3H]Met-hexanucleotide is also soluble in ethyl acetate. The assay is sensitive to 0.05 pg of RNase A, has a very low background, and is linear with enzyme concentration. The most economic substrate is unfractionated tRNA containing a small concentration of the radiolabeled aminoacyl-tRNA.

Acetates↗