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M Staehelin

Publications and source records attributed to M Staehelin.

At least 37 records · Page 2Linked to original sources
PubMed1976

An improved method for the separation and quantitation of the modified nucleosides of transfer RNA.

A method is described which allows a very efficient determination of the modified nucleosides of tRNA. The technique involves enzymatic degradation of the tRNA to nucleosides at pH 7.6 and their separation by two-dimensional thin-layer chromatography on cellulose-coated aluminum foils. Based on the analysis of two mammalian tRNAs it is shown that the technique is suitable for the determination of chemically unstable nucleosides as well as the ribose-methylated compounds. At least 36 of the 45 known modified nucleosides can be separated and quantitatively determined by the method described. This procedure is especially suitable for the estimation of the nucleoside composition of unlabeled tRNAs as well as for studying the post-transcriptional modifications of tRNA.

Aluminum↗
PubMed1975

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.

Adenosine Triphosphatases↗
PubMed1974

N2-guanine specific transfer RNA methyltransferase I from rat liver and leukemic rat spleen.

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N(2)-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m(2)G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA(Arg), tRNA(Phe), and tRNA(Val) yielded significantly more m(2)G than the bulk tRNA. The K(m) for tRNA(Arg) in the methylation reaction with enzymes from either tissue was 7.8 x 10(-7) M as compared to the value 1 x 10(-5) M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA(Arg), tRNA(Phe), and tRNA(Val), A-m(2)G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5'-end contained in a sequence (s(4))U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.

Animals↗
PubMed1974

N2-guanine specific transfer RNA methyltransferase II from rat liver.

N(2)-guanine methyltransferase II was purified from rat liver. This enzyme methylated bulk E. coli tRNA to an extent of 7.6 nmoles of methyl groups/mg tRNA. Oligonucleotide analysis showed that N(2)-methylated guanosines were present in the modified tRNA in two sequences, namely Y-m(2)G-Cp and Y-m(2) (2)G-Cp in the ratio 4:3. Two pure tRNA(Leu) species, and tRNA(Met) (f) from E. coli were methylated with the enzyme to extents of 17, 11, and 8 nmoles of methyl groups incorporated per mg tRNA, respectively. When the methylated tRNAs were analysed no m(2) (2)G was detected and the m(2)G occurred in the tRNAs specific for leucine in a Y-m(2)G-Cp sequence and in the tRNA(Met) (f) in a sequence Y-m(2)G-Up.It is concluded that the mammalian enzyme specifically recognizes the interstem unpaired guanylate residue between the dihydrouridine arm and the anticodon arm. The absence of any detectable m(2) (2)G methylation of individual tRNA species is discussed.

Animals↗