Loss Of Acceptor Activity Research Articles

Functional assay of tRNA molecules transcribed from a purified gene.

Purified tRNA genes are expressed when microinjected into the nucleus of X.laevis oocytes. In this paper we describe a method to assay the capacity to be aminoacylated of the tRNA transcribed in the frog oocytes. The method exploits the radiochemical purity of the transcript and relies on the binding of aminoacyl-tRNA but not of uncharged tRNA to purified elongation factor EF-Tu. We also present some preliminary results on several single point mutants of tRNAPro from Caenorhabditis elegans. We show that nucleotide 73 of tRNAPro can be substituted by any other nucleotide without loss of acceptor activity. A double mutant, causing transition from G45G46 to A45A46 has lost acceptor activity. Also inactive is a mutant carrying an insertion of a single base in the anticodon loop.

Lysosomal enzyme targeting. N-Acetylglucosaminylphosphotransferase selectively phosphorylates native lysosomal enzymes.

Lysosomal enzymes contain 6-phosphomannosyl moieties which mediate their translocation to lysosomes. This recognition marker is synthesized by the sequential action of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase. A new assay for the N-acetylglucosaminylphosphotransferase, using alpha-methylmannoside as acceptor, is presented. Using this assay, we partially purified the transferase and examined its substrate specificity. The transferase exhibited a very high affinity toward lysosomal enzymes (apparent Km values of less than 20 microM) and was greater than 100-fold more efficient (Vmax/Km) when using lysosomal enzymes as acceptors as compared to nonlysosomal glycoproteins that contain high mannose oligosaccharide units. Heat denaturation of the lysosomal enzymes resulted in the loss of acceptor activity. The model compounds alpha-methylmannoside and Man5--8GlcNAc were poor acceptors. We propose that this enzyme catalyzes the initial, determining step by which synthesized acid hydrolases are distinguished from other newly synthesized glycoproteins and thus are eventually targeted to lysosomes.

Temperature dependence of the aminoacylation of tRNA by Bacillus stearothermophilus aminoacyl-tRNA synthetases.

AbstractValine specific transfer RNA (tRNAVal) was isolated from Bacillus stearothermophilus and Escherichia coli by chromatography on benzoylated DEAE–cellulose (BD–cellulose). Likewise isoleucine specific transfer RNA (tRNAIle) was isolated from B. stearothermophilus and from Mycoplasma sp. Kid. The thermal denaturation profiles (melting curves) of the two tRNAVal species in the presence of Mg+ + were nearly identical. However, the Tm for the Kid tRNAIle was about 10°C lower than that for the B. stearothermophilus tRNAIle. A nuclease and tRNA‐free aminoacyl‐tRNA synthetase (AA‐tRNA synthetase) preparation from B. stearothermophilus was able to function efficiently at temperatures up to 80°C in the aminoacylation of all four tRNA species. Determination of the amino acid‐acceptor activity of each tRNA species as a function of temperature of the aminoacylation reaction showed in each case a strong correlation between the loss of acceptor activity and the thermal denaturation profile of the tRNA. Evidence is presented that the loss in acceptor activity is most likely due to a change in structure of the tRNA as opposed to denaturation of the enzyme. These results further support the idea that correct secondary and/or tertiary structure must be maintained for tRNA to be active as a substrate for the AA‐tRNA synthetase.