Enzymes differential expression

Dangel W, R Brackmann, A Lack, M Mohamed, J Koch, J Oswald, B Seyfried, A Tschech, G Fnchs (1991) Differential expression of enzyme activities initiating anoxic metabolism of varions aromatic compounds via benzoyl-CoA. Arch Microbiol 155 256-262. 

These enzymes (and transporters) exhibit differential expression at various sites throughout the GIT. For example, CYP3A4 expression is highest in the duodenum and lowest in the colon conversely, the expression of P-gp is greatest in the colon. This has implications for the gut wall first-pass extraction of drugs delivered by modified-release formulations, where the majority of the drug must be absorbed from the colon. 

Capsaicinoids are synthesized by the condensation of vanillylamine with a short chain branched fatty acyl CoA. A schematic of this pathway is presented in Fig. 8.4. Evidence to support this pathway includes radiotracer studies, determination of enzyme activities, and the abundance of intermediates as a function of fruit development [51, 52, 57-63], Differential expression approaches have been used to isolate cDNA forms of biosynthetic genes [64-66], As this approach worked to corroborate several steps on the pathway, Mazourek et al. [67] used Arabidopsis sequences to design primers to clone the missing steps from a cDNA library. They have expanded the schema to include the biosynthesis of the key precursors phenylalanine and leucine, valine and isoleucine. Prior to this study it was not clear how the vanillin was produced, and thus the identification of candidate transcripts on the lignin pathway for the conversion of coumarate to feruloyl-CoA and the subsequent conversion to vanillin provide key tools to further test this proposed pathway. 

These examples demonstrate that both genetic and environmental factors are involved in determining the activity of this and other cytochrome enzymes. Distinct patterns of genetic polymorphisms exist across ethnic groups and these can be tested and investigated alongside possible environmental and dietary factors that may cause differential expression of these genes. 

Physiological optimization of enzyme synthesis by variation of the culture parameters is usually required to enhance the catalytic activity of whole-cell biocatalysts to such a level that it can be apphed in a biocatalytic process. In addition, physiological conditions can influence the selectivity of the reaction, since enzymes with opposite selectivities can be differentially expressed. In some cases, genetic engineering is required to obtain biocatalysts with a desired selectivity that does not consume the product of choice (see 5.3.5). Alternatively, one may choose to isolate the desired activity from the culture in order to use the biocatalyst in an enzyme reactor. 

CYP2E1, an enzyme catalysing vinylidene chloride activation, is expressed in various human tissues, including liver, although functional evidence is lacking for many tissues. The level expressed in human liver is similar to that in rodents. CYP2E1 seems to be differentially expressed in kidney microsomes from humans (no expression), rats (no expression) and mice (high expression in males). 

Efforts to elucidate the genetic basis of biosynthetic pathways tend to result in the identification of structural genes, i.e. genes encoding enzymes that catalyze the conversion of intermediates in a specific pathway. This is in part due to the relatively high expression of the structural genes, which results in abundant representation in expressed sequence tag (EST) libraries, or high probability of identification when cDNA subtraction methods or other methods that rely on differential expression are used. 

Brady, C.P., Brindley, P.J., Dowd, A.J. and Dalton, J.P. (2000a) Schistosoma mansoni differential expression of cathepsin L1 and cathepsin L2 suggests discrete biological functions for each enzyme. Experimental Parasitology 94, 75-83. 

The second messenger molecules Ca2+ and cyclic AMP (cAMP) provide major routes for controlling cellular functions. In many instances, calcium (Ca2+) achieves its intracellular effects by binding to the receptor protein calmodulin. Calmodulin has the ability to associate with and modulate different proteins in a Ca2+-dependent and reversible manner. Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE, EC 3.1.4.17) is one of the key enzymes involved in the complex interactions that occur between the cyclic-nucleotide and Ca2+ second messenger systems (see Figure 13.2). CaMPDE exists in different isozymic forms, which exhibit distinct molecular and catalytic properties. The differential expression and regulation of individual phosphodiesterase (PDE) isoenzymes in different tissues relates to their function in the body. 

The biochemistry of odor detection involves at least three types of protein odor receptors (ORs) odorant binding proteins (OBPs) and odor degrading enzymes (ODEs). ORs are expressed by olfactory receptor neurons (ORNs) and localized in the membranes of the ciliated dendrites (Figure 14.1). The result of detection is translated into neuronal electrical activity by transductory proteins. But while transductory proteins are more or less common for all olfactory neurons, differential expression of ORs, OBPs and ODEs allows the neurons to detect specific odor molecules. 

Twenty-two top-ranking reactions of the parasite whose enzymes were significantly differentially expressed under drug (tetracycline) influence vs. their expression under no drug influence... 

Three forms of branching enzyme from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized.260 Two forms are immunologically related to maize BEI and one form to maize BEII. The N-terminal sequences are consistent with these relationships. The wheat BEIb gene is located on chromosome 7B, while the wheat BEIad peptide genes are located on chromosomes 7 A and 7D. The BE classes in wheat are differentially expressed during endosperm development. BEII is constitutively expressed throughout the whole cycle, while BEIb and BEIad are only expressed in late endosperm development. 

The multienzymic nature of pyruvate kinase has been investigated in detail in M. expansa (98), H. diminuta (117) and S. erinacei (240). These worms possess FBP-sensitive and FBP-insensitive pyruvate kinase isoenzymes. In H. diminuta, as many as five pyruvate kinase isoenzymes (for definition and usefulness, see Chapter 6) occur during development (Fig. 5.3) and it seems likely that differential expression of these different forms of the enzyme may help to control the specific composition of excreted end-products by the various life cycle stages. The nature and regulation of the end-products secreted in H. diminuta are discussed further below. 

In the past, various serendipitous discoveries have capitalized on the differential expression of enzymes by host and viral infected cells. For example, the prodrug Acyclovir, used widely for the treatment of herpes simplex and herpes zoster infections, is selectively activated through phosphorylation by viral thymidine kinase to acyclovir monophosphate which is then converted to the triphosphate, which inhibits DNA polymerase, by host cellular enzymes. Similarly several 2, 3 -dideoxynucleoside analogs such as Zidovudine (azidothymidine, AZT) and 2, 3 -didehydro-3 -deoxythymidine (D4T) have potent antiviral activity against human immunodeficiency vims (HIV). These compounds are selectively phosphoiylated intracellularly to the 5 -triphosphate derivatives which inhibit the viral reverse transcriptase. 

The presence of the second active form of MIPS with a —65 kDa subunit indicated that the inositol requirement of the organism might be provided by the interplay of two different MIPS enzymes, probably by differential expression through time and space. The results indicate that the —65 kDa MIPS protein of Synechocystis might be coded by the ORF sill981, annotated as a putative acetolactate synthase (unpublished data from this laboratory). This gene also functionally complements the inositol auxotrophic yeast strain FY250 and Schizosaccharomyces pombe, a natural inositol auxotroph, and the expressed protein is immunoreactive to anti MIPS antibody. 

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