Amino acids in vitro

Initial attempts at introducing unnatural amino acids into proteins in mammalian cells involved the transfection of an amber suppressor tRNA that is chemically acylated with an unnatural amino acid in vitro This approach limits the amount of overall protein that can be produced since the acylated suppressor tRNA is consumed stoichiometrically and cannot be regenerated inside cells. It is also technically demanding to prepare the chemically acylated tRNA. 

Navab, F. and Winter, C. G., Effect of aging on intestinal absorption of aromatic amino acids in vitro in the rat, Am. ]. Physiol., 254, G631, 1988. 

Several strategies exist for incorporating noncanonical amino acids into proteins.One powerfiil approach uses stop, or nonsense, codons that normally terminate protein translation. Suppressor tRNAs recognize stop codons and suppress translational termination. By linking a noncanonical amino acid to a suppressor tRNA by chemical misacylation, one can insert the new amino acid in vitro in response to a stop codon. " To accomplish the same objective in vivo, however, an aminoacyl-tRNA synthetase that recognizes and couples the suppressor and the noncanonical amino acid must be developed. This method enables insertion of a single noncanonical amino acid at a specified position in a protein. 

Sandberg M, Jacobson I, and Hamberger A. (1982) Release of endogenous amino acids in vitro from the colliculus and the hippocampus... 

Zhou,J., Ertel, S.I., Buettner, H.M. and Kohn,J. (1994) Evaluation of tyrosine-derived pseudo-poly(amino acids) In vitro cell interactions. 20th Annual Meeting of the Society for Biomaterials. Boston MA, Society for... 

Elsenhans, B., Sufke, V., Blume, R., and Caspary, W. F., 1980, The influence of carbohydrate gelling agents on rat intestinal transport of monosaccharides and neutral amino acids in vitro. Clin. Sci. 59 373. 

Low dosages of trichothecenes inhiiit membrane transfer of gkjcosr, ciddum, and some amino acids in vitro. These effects are indeperxlent of the inhibition of protein synthesis. 

Signification of results with isolated mitochondria. The site of incorporation of radioactive amino acids in vitro is almost exclusively insoluble membrane-bound protein (structural protein(s)) while soluble proteins, i.e. those readily lost from mitochondria are unlabelled. A comparable situation is observed with isolated chloroplasts. The comparison of the labelling of the mitochondrial substructures after careful fractionation shows that it is predominantly the inner membrane which is active in protein synthesis. It is concluded that soluble enzymes (cytochrome c, dehydrogenases are easily dissolved) and the outer mitochondrial membranes are synthesized outside of the mitochondria in vivo. 

The authors suggested that ethylene imine formed active secondary mutagens, connected with the components of the cell, including nucleic acids. In the third article of this series [21], the authors added ethylene imine to amino acids in vitro glycine and histidine, to the hexamine (hexamethylenetetramine), to vitamins thiamin (vitamin Bj), nicotinic acid. 

Klein and Olsen126 studied the action of kojic acid on the enzymic oxidation of amino acids by the liver and kidney of rats. Low concentrations of kojic acid in vitro inhibited the oxidation of a number of D-amino acids, L-phenylalanine, and a few related compounds. Kojic acid was found to compete with D-amino acid oxidase for the substrate. 

Subsequently, serotonin was shown to affect responses to excitatory amino acids in rat neocortical neurons, in cells of the ventrobasal thalamus, dorsal horn neurons, and rat locus coeruleus neurons (Aston-Jones et al. 1991 S. A. Eaton and Salt 1989 Murase et al. 1990 Read et al. 1990 J. N. Reynolds et al. 1988]. Whether the modulatory effect of serotonin is to enhance or attenuate the effect of the excitatory amino acid appears to depend on the brain region investigated. Also, many of the experiments used extracellular recording techniques, although Sizer et al. (1992] used intracellular current clamp in vitro experiments to investigate neurons of the rat entorhinal cortex and neocortex. In the entorhinal cortex and neocortex, the predominant effects of serotonin were to reduce and enhance, respectively, the response to excitatory amino acids. 

Dye, D.J., and Taberner, P.V. The effects of some newer anaesthetics on the in vitro activity of glutamate decarboxylase and GABA transminase in crude brain extracts and on the levels of amino acids in vivo. J. Neurochem. 24 997-1001, 1975. 

Borsook, H. Abrams, A. Lowy, P. H. Fructose-amino acids in liver stimuli of amino acid incorporation iri vitro. J. Biol. Chem. 1955, 215, 111-24 through ref. 72. 

An example of using the combinatorial approach to drug discovery is the attempt to produce a miniprotein version of erythropoietin (EPO). As mentioned previously, EPO stimulates the body to produce red blood cells. EPO is widely used for patients with anemia, kidney disease, cancer, or AIDS and has yearly sales of approximately 1 billion. By applying combinatorial chemistry, a new 20-amino-acid molecule was discovered that successfully mimics the 165-amino-acid EPO, in vitro. Of particular interest is the fact that the sequence of amino acids in the small molecule is unrelated to that of the larger EPO. This research also suggests that it may be possible to make nonprotein versions of other important proteins such as insulin and avoid the need for injections. 

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