PubMed · 1371726
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T Delahunty. 1992. Hemoglobin variants.. https://pubmed.ncbi.nlm.nih.gov/1371726/
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Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.
Glycosylation is a major pathway for posttranslational modification of tissue protein and begins with nonenzymatic addition of carbohydrate to the primary amino groups. Excessive glycation of tissue protein has been implicated in the pathogenesis of diabetes and ageing. While glycation of aminophospholipids has also been postulated, glycated aminophospholipids have not been isolated. Using normal phase HPLC with on-line electrospray mass spectrometry we found glycated ethanolamine phospholipids to make up 10-16% of the total phosphatidylethanolamine (PE) of the red blood cells and plasma of the diabetic subjects. The corresponding values for glycated PE of control subjects were 1-2%.
Evidence is presented for some secondary structure, very likely alpha-helical, of the propeptide of subtilisin E in aqueous salt solution, as well as for strong intermolecular interactions between the propeptide and the mature sequence both in the processed and unprocessed states (i.e. in prosubtilisin). Prosubtilisin is shown to exist as a dimer according to size exclusion high performance liquid chromatography under nondenaturing conditions; that dimer may be on the autoprocessing pathway. According to such a model, the prosequence of one prosubtilisin molecule is the template for the refolding of the mature sequence of the second, and, in turn, the hydrolytic process is intermolecular as well. Support for such an intermolecular folding model also includes potent slow binding inhibition of subtilisin by the propeptide, specific proteolysis of the propeptide by subtilisin, and evidence for intermolecular processing under a variety of conditions.