Isozymes, amylases

Drought also has a profound effect on protein synthesis. In many plant tissues, a reduced water potential causes a reduction of total protein synthesis and a rapid dissociation of polyribosomes. The latter has been shown not to be the consequence of increase in ribonuclease activity (Hsiao, 1973 Dhindsa Bewley, 1976). For a specific protein, Jacobsen, Hanson Chandler (1986) have shown in barley leaves that water stress enhances the synthesis of one of the a-amylase isozymes. Using a cDNA probe they found that water-stressed leaves contained much more a-amylase mRNA than unstressed plants. 

As indicated in Table 2.1, most of the promoters used in plant tissue culture have been based on the constitutive cauliflower mosaic virus (CaMV) 35S promoter. In contrast, inducible promoters have the advantage of allowing foreign proteins to be expressed at a time that is most conducive to protein accumulation and stability. Although a considerable number of inducible promoters has been developed and used in plant culture applications, e.g. [32-37], the only one to be applied thus far for the production of biopharmaceutical proteins is the rice a-amylase promoter. This promoter controls the production of an a-amylase isozyme that is one of the most abundant proteins secreted from cultured rice cells after sucrose starvation. The rice a-amylase promoter has been used for expression of hGM-CSF [10], aranti-trypsin [12, 29, 38, 39] and human lysozyme [30]. 

Enzymes that perform the same catalytic function are known as homologous enzymes and fall into two classes. Heteroenzymes are derived from different sources and although they catalyse the same reaction they show different physical and kinetic characteristics. The hydrolytic enzyme a-amylase (EC 3.2.1.1) is found in the pancreatic secretion in man and is different from the enzymes of the same name which are derived from bacteria or malt. Iso-enzymes, sometimes referred to as isozymes, are different molecular forms of the same enzyme and are found in the same animal or organism although they often show a pattern of distribution between tissues. 

Pancreatic isozymes. In AP there is a significant increase in molecular forms P2 (approximately 91% of patients) and P3 (approximately 98% of patients) (P2, W5) as well as the presence of the isozyme P4, which does not appear in healthy controls (P2, N2). In the presence of a cyst, the P4 and P5 isozyme fractions are increased to >15% of total amylase activity. In the absence of a cyst, the P4 and P5 fractions make up <15% of total enzyme activity (T6). 

Ainsworth, C.C., Gale, M.D., and Baird, S. 1983. The genetic of (3-amylase isozymes in wheat. Allelic variation among hexaploid varieties and intrachromsomal gene locatios. Theor Appl Genet 66(1) 39 -9. 

The two-dimensional western blot analysis shows numerous isozymes or isoforms in both M202 seed and shoot callus, several of which don t appear in the germinating seed. Evidently in the callus there is a derepression or stimulation of many, if not most, of the a-amylase genes. Some of the spots on the western blot may be due to charge modifications of some isozymes, and therefore do not represent products of separate genes. 

Little is known of the structure, the nature of the active site, or the physical properties of the glucoamylases. The purified enzyme firom A. niger has been reported to contain at least two isozymes, which are readily separated by electrophoresis, - and gommc-amylase has been separated into two components, one of which has an optimal pH of 4, and the other, of about 6.5. 

Hokari, S., Miura, K., Koyama, 1., Kohayashi, M., Matsunaga, T., lino, N., and Komoda, T. (2003) Expression of alpha-amylase isozymes in rat tissues. Comp. Biochem. Physiol. B Biochem. Mol Biol, 135, 63-69. 

Evidence has been accumulated over a number of years to show that there is more than one a-amylase involved in starch digestion. Seven isozymes of a-amylase appear in GA-treated isolated aleurone layers and all appear to be of approximately the same molecular weight (MW 45,000) [28]. 

In Chapter 6 it was pointed out that there are several isozymes of a-amylase — according to recent work, as many as seven. In earlier studies, where only four isozymes were isolated [61], all four were found to be synthesized de novo in GA-treated aleurone layers. Whether the other three isozymes arise de novo... 

According to the data mentioned above, the organization of the block which contains esterase genes can be schematized as in Fig. 33 B. This scheme explains the rare appearance of individuals in some strains, who displayed an equal expression of the 4S and 4F subfractions. The existence of 4S and 4F fractions with similar (both intensive and weak) activity expressions could be a consequence of the crossing-over between corresponding, closely linked genes (Fig. 33. B). A similar mechanism was described for the amylase isozymes in D. melanogaster (Bahn, 1967). 

The influence of positional effect on the phenotypic expression of isozymes was also demonstrated in the case of amylase from D. melanogaster (Bahn, 1971). 

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