Kerstin Kjellin

STUDIES ON MICROBIAL RNA. I. TRANSFER OF METHYL GROUPS FROM METHIONINE TO SOLUBLE RNA FROM ESCHERICHIA COLI.

Two partially purified enzyme preparations, one from yeast and one from Escherichia coli, have been used to study the transfer of methyl groups from methionine to soluble RNA (S-RNA) from E. coli. The reaction requires ATP and magnesium. The E. coli enzyme can incorporate methyl groups only into S-RNA from methionine-starved E. coli K12 W6 (a mutant with a deficient control of RNA synthesis). The yeast enzyme can methylate S-RNA from E. coli K12 in log phase as well as from methionine-starved cultures of W6. The methylation is not inhibited by periodate oxidation of the terminal adenosine residue in the S-RNA. The alkaline stability of the methylated S-RNA is clearly different from that of methionyl RNA. Methylated S-RNA has been degraded by snake venom enzymes as well as by alkaline hydrolysis. The digestion products have been examined by paper and anion exchange chromatography, respectively. In vitro, neither of the two enzymes can methylate purified ribosomal RNA (R-RNA). In vivo incorporation of methionine has, however, shown a significant methylation of both S-RNA and R-RNA. The composition of the methylated nucleosides obtained from R-RNA differs from that of S-RNA. Total RNA contains also smaller amounts of non-methylated RNA. Counter-current distribution of S-RNA has shown that the acceptors for the methyl groups can be separated from the acceptors for tyrosine.

A rapid semi-continuous method for purification of ceruloplasmin from human serum☆

Abstract A three-step method for ceruloplasmin purification has been devised, which operates throughout under mild conditions (4°, potassium phosphate buffer (pH 6.8), with KCl to stabilize ceruloplasmin). Steps 1 and 3 utilize protein-protein displacement on DEAE-Sephadex and hydroxyapatite, respectively, while the second step consists of gel-filtration on Sephadex G-100. Insufficiently purified ceruloplasmin fractions obtained at various stages are diluted to the appropriate ionic strength and re-utilized. After Step 2 the ratio of the absorbancies at 610 and 280 mμ was 0.040, which corresponds with a degree of purification of about 95%. After Step 3 the ratio increased to 0.042, corresponding to practically 100% pure ceruloplasmin. The overall recovery was about 85%, when recycling of impure material was included. One complete cycle takes 2.5 days and gives about 200 mg of pure material. A new cycle can be started once a day.