Experiments with highly purified amino acid

Experiments With Highly Purified Amino Acid Carrier Preparations... 

McCoy RH, Meyer CE, Rose WC. 1935. Feeding experiments with mixtures of highly purified amino acids. Vlll. Isolation and identification of a new essential amino acid. J Biol Chem 112 283-302. (Reports the discovery of the final lAA, threonine)... 

The nature of the active site in beta-amylase is not unambiguously known for enzymes from different sources. Early experiments on purified barley and on malted barley first indicated, from studies of the modification of the enzyme with nitrous acid and ketene, that free tyrosine and sulfhydryl groups are essential for activity, whereas free a-amino groups are not. The importance of the sulfhydryl groups was emphasized by the partial recovery of activity of the modified or oxidized enzyme (that is, treated with nitrous acid, iodine, phenyl mercuribenzoate, ferricyanide, and cupric ions) when it was treated with hydrogen sulfide or cysteine. Barley feeto-amylase (not highly purified) has been reported to contain 12—15 sulfhydryl groups per molecule by titration with p-chloromercuribenzoate, and the loss of free sulfhydryl content by treatment with L-ascorbic acid in the presence of cupric ions was found to be directly related to the loss of activity. 

Recently Hansson and Wikvall studied 12 -hydroxylation in a reconstituted system consisting of highly purified cytochrome P-450 LM4 from rabbits [101]. The cytochrome P-450 fractions used were electrophoretically homogenous, but the cytochrome P-450 from starved rabbits had up to 4 times higher capacity to catalyse 12a-hydroxylation than had cytochrome P-450 from untreated, phenobarbital-treated, or -naphthoflavone-treated rabbits. It should be mentioned that treatment with /8-naphthoflavone increases the amount of cytochrome P-450 LM4 in the liver. Amino acid analyses, peptide-mapping experiments as well as absorption spectral and circular dichroism spectral analyses revealed physical differences between cytochrome P-450 LM4 preparations from starved and phenobarbital-treated animals. It was concluded that the cytochrome P-450 LM4 fraction was heterogenous and contained a species of cytochrome P-450 specific for 12a-hydroxylation. 

Theoretically, one should be able to make as long a peptide as desired with this technique. Reactions do not produce 100% yields, however, and the yields are further decreased during the purification process. After each step of the synthesis, the peptide must be purified to prevent subsequent unwanted reactions with leftover reagents. Assuming that each amino acid can be added to the growing end of the peptide chain in an 80% yield (a relatively high yield, as you can probably appreciate from your own experience in the laboratory), the overall yield of a nonapeptide such as bradykinin would be only 17%. It is clear that large polypeptides could never be synthesized in this way. 

A number of studies have shown that natural metabolites can inhibit transamination. With a partially purified mung bean preparation which could use lysine, methionine, or aromatic amino acids as amino donors, it was found that the aliphatic substrates (e.g., lysine and methionine) inhibited the transamination of phenylalanine. The extent of this inhibition was related to their effectiveness as substrates, suggesting that they competed with phenylalanine (Gamborg, 1965). Using the highly purified but multispecific aromatic amino acid (and aspartate) aminotransferase from bush bean. Forest and Wightman (1973) demonstrated that 40 mM aspartate inhibited transamination of L-phenylalanine (40 mM) by 85%. Further experiments showed that elevated concentrations of phenylalanine reduced the inhibition by aspartate double-reciprocal plots indicated competitive inhibition. These... 

Until recently, this has been one of the most useful of the isotope dilution methods because it allows the quantitative conversion of a small amount of an unlabelled compound to an isotopically labelled derivative. The unlabelled compound is reacted quantitatively with a radioactive agent of known specific activity and the radioactive derivative is isolated, purified and counted. The activity recorded indicates the amount of radioactive reagent it contains and since the stoichiometry of the reaction is known the amount of compound present can be calculated. There are two basic requirements. First, the compound to be analysed must be quantitatively converted in high and reproducible yield to the radioactive derivative second, there must be a good method of isolating and purifying the labelled derivative. The first experiments to use an isotope derivative technique focused on the determination of amino acids in a mixture [263-265]. The reagent used was I-pipsyl chloride (/ -iodobenzenesulphonyl chloride). 

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