Rough enzyme characterization

When the appropriate enzyme reaction was detected, a rough enzyme characterization was performed including determination of the molecular mass, pH optimum, isoelectric point, preliniinary substrate acceptance (substrate specificity), and cofactor studies. 

Nitrile hydratase was initially discovered by Yamada and co-workers of our laboratory [46]. The enzyme that catalyzes the conversion of nitriles into amides is clearly distinguishable from nitrilase in the mode of nitrile-degradation. Thus, the enzyme was termed nitrile hydratase (NHase) . This enzyme was purified and characterized from various micro-organisms. NHases are roughly classified into two groups according to the metal involved Fe-type and Co-type [45],... 

Nitrilase was initially discovered in plants as an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) [74,75], Recently, four genes of nitrilases (belonging to arylacetonitrilase) involved in the IAA biosynthesis have been cloned and characterized from Arabidopsis thaliana [76-78], After the discovery of the plant nitrilase in 1964, various nitrilases were purified and characterized [41], Nitrilases are roughly classified into three major categories according to substrate specificity (i) aromatic nitrilase, which acts on aromatic or heterocyclic nitriles (ii) aliphatic nitrilase, which acts on aliphatic nitriles (iii) arylacetonitrilase, which acts on arylacetonitriles. These three types... 

All SABATH proteins characterized so far contain between 357 and 389 amino acids, and the molecular mass of the subunit ranges from 40-49 kDa. Size exclusion chromatography, performed with some of these enzymes, has revealed that the molecular mass of the native enzymes is roughly 80 kDa, indicating a dimeric structure. None of the SABATH proteins identified thus far have any obvious subcellular targeting signals. Cytosolic localization was experimentally confirmed for two of these enzymes, BAMT and CaMXMT.57,59... 

O-methylation of 16-hydroxytabersonine is followed by an uncharacterized hydration step, and then by TV-methylation of the N-indole by the enzyme S -adenosyl - L-methionine 16-methoxy - 2,3-dihydro - 3-hydroxytabersonine - IV-methyltransferase (NMT), originating desacetoxyvindoline, Fig. (6). NMT has been roughly characterized by DeLuca et al. [95], and partially purified by Dethier and DeLuca [96] from young leaves of 6 months old C. roseus plants. Subcellular localization studies indicated that NMT is specifically associated with the membranes of thylakoids [46]. 

The flux of a substrate (rate of transport) across the plasma membrane via transporter-mediated processes is characterized by saturabUity. The relationship between the flux v and substrate concentration C in a transporter-mediated process is analogous to the rate of product formed by an enzyme and the concentration of substrate. The maximum transport rate is proportional to the density of transporters on the plasma membrane, and the represents the substrate concentration at which the flux is half maximal. When C is small compared with the K, the flux is increased in proportion to the substrate concentration (roughly linearly). If C is large compared with the K value, the flux approaches the maximal value (V ). The and V values can be deter-... 

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