Aspartate aminotransferase, preparation

Fig. (5). Effects of Some OGs on Primary Cultured Rat Hepatocytes injured with CC14 Effects of glycyrrhizin (GL, triangles), kaikasaponin III (2 1, Kaika III, circles) and soyasaponin I (1, Soya I, squares) on CCI4 (SmM)-induced cytotoxicity in primary cultured rat hepatocytes. The marker of liver injury is the aspartate aminotransferase (AST), measured in IU (International Units)/ . Data are the mean S.D. for three independent cell preparations.

The aminotransferase class of enzymes (E.C. 2.6.1.x), also known as transaminases, are ubiquitous, PLP-requiring enzymes that have been used extensively to prepare natural L-amino acids and other chiral compounds.30 123 124 The L-aminotransferases catalyze the general reaction shown in Scheme 19.19 where an amino group from one L-amino acid is transferred to an a-keto acid to produce a new L-amino acid and the respective a-keto acid (see also Chapter 3). Those enzymes most commonly used as industrial biocatalysts have been cloned, overexpressed, and generally used as whole-cell or immobilized preparations. These include branched chain aminotransferase (BCAT) (E.C. 2.6.1.42), aspartate aminotransferase (AAT) (E.C. 2.6.1.1), and tyrosine aminotransferase (TAT) (E.C. 2.6.1.5). 

Vinylglycine is a natural amino acid found in mushrooms. It is an inhibitor of pyridoxai-linked aspartate aminotransferase, and has also been postulated as an intermediate in the enzymatic conversion of homoserine to threonine and o-ketobutyrate. Protected vinylglycine is also a versatile asymmetric starting material for synthesis.7 Variants have been prepared in racemic. " optically active, optically pure,2.15-17 and isotopically labeled form.4b.i8-20 xhis procedure is derived from our earlier publication and contains improvements in procedure and scale-up. 

The requirements for, preparation of, and application of enzyme reference materials have been discussed extensively (134, 135, 151, 152, 153, 154, 155, 156, 157, 158). Cooperation among clinical enzvmologists in Europe and the United States over the last several years has resulted in the availability of only a few enzyme reference materials. SRM 8430 from NIST is a preparation of human erythrocyte aspartate aminotransferase in a human albumin matrix (144) certified reference material (CRM) 319 from the Community Bureau of Reference (BCR) of the Commission of the European Communities is a preparation of porcine y-glutamvltransferase in a bovine serum matrix (159). The working group of the BCR is in the process of establishing protocols and evaluating... 

Immunological methods for enzymes, more specifically isoenzymes, such as lactate dehydrogenase-1 (167, 168), mitochondrial aspartate aminotransferase (169), prostatic acid phosphatase (170, 171,172), and creatine kinase-MB (173, 174, 175), have been in use in the clinical laboratory for 10 years. However, the use of the immunological rather than catalytic properties of enzymes has not provided the opportunities for standardization that was anticipated a number of years ago (176, 177, 178). It is only within the last year that a working group on CK-MB mass assay was formed under the auspices of the Standards Committee of the American Association for Clinical Chemistry (AACC). The objective of this working group is to prepare a reference material to calibrate methods that are based on the principle of CK-MB mass measurement. 

Confirmation of the molecular structure of the enzyme-inactivator adduct has been obtained for few modified PLP-dependent enzymes. In the case of the reaction of aspartate transaminase (aspartate aminotransferase) with L-serine 0-sulfate, the surprising result thus obtained by Metzler and co-workers has forced reevaluation of the mechanism of similar inactivators (Ueno et al., 1982). Conventional wisdom argued that the reaction should involve elimination of sulfate from the inactivator followed by addition of an enzyme nucleophile to the resulting double bond (Fig. 8). When subjected to high pH, however, the inactivated enzyme releases a yellow PLP adduct which has been identified as the aldol product of the cofactor and C-3 of pyruvate (9, Fig. 9) as previously prepared by... 

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 now it has not been elucidated what is the origin of mitochondrial sulphurtransferases, although majority of mitochondrial proteins is synthesized on cytoplasmic ribosomes and later transferred to the organelle /for references see Schatz and Mason, 197 / Recently Marra and co-workers /1978/ studied selective permeation of labelled aspartate aminotransferase isoenzymes into mitochondria in vitro. Following their ideas.we labelled crystalline bovine liver rhodanese with and Incubated it with fresh preparation of... 

The synthesis of chiral a-amino acids starting from a-keto acids by means of a transamination has been reported by NSC Technologies [26, 27]. In this process, which can be used for the preparation of l- as well as D-amino acids, an amino group is transferred from an inexpensive amino donor, e.g., L-glutamic acid, l-22, or L-aspartic acid, in the presence of a transaminase (= aminotransferase). This reaction requires a cofactor, most commonly pyridoxal phosphate, which is bound to the transaminase. The substrate specificity is broad, allowing the conversion of numerous keto acid substrates under formation of the L-amino acid products with high enantioselectivities [28]. 

A racemase brings about inversion of the relatively cheap (L)-isomers of alanine or aspartic acid, but not of (D)-phenylalanine. Only (L)-phenylalanine is deaminated by an (L)-amino acid deaminase, whereas (D)-phenylalanme is not. The latter is generated by ammonia transfer from (D)-alanine or (D)-aspar-tic acid with a (D)-amino acid aminotransferase. The equilibria are moved in favour of the product, either by the metabolism of pyruvic acid or oxosuccinic acid. Since (L)-amino acid deaminases, like (D)-amino add aminotransferases, are non-specific, they also permit the preparation of a variety of other (D)-amino acids. [58]... 

S)-Amino-3-[3- 6-(2-methylphenyl) pyridyl]-propionic add 82a was prepared by an enzymatic deracemization process using a combination of two enzymes (P)-amino acid oxidase from Trigonopsis variabilis cloned and expressed in E. coli and an (S)-aminotransferase from Sporosarcina ureae, also cloned and expressed in E. coli [147]. Racemic amino acid 82 was used as a substrate and (S)-aspartate was used as amino donor. An (S)-aminotransferase was also purified from a soil organism identified as Burkholderia sp. and cloned and expressed in E. coli and used in this process [147]. This process was scaled up to 70 L scale. 

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