Enzyme Complementation

Another technology for the study of protein-protein interactions in cells is based on intracistronic enzyme complementation. 3-galactosidase is the most frequently used 

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Microsomes Can be frozen and used on demand Contains major drug metabolizing enzymes Straighforward to use Limited enzyme complement Closed system so may not be representative of in vivo situation... 

Hepatocytes Culture methods available Full enzyme complement - contain enzymes and enzyme cofactors not present in subcellular fractions Full enzyme complement - contain enzymes and enzyme cofactors not present in subcellular fractions Can be used quantitatively Useful for prediction of Phase 1 and 11 metabolism Require fresh tissue No cell-cell contact Need collagenase digestion Handling difficult Closed system so may not be representative of in vivo situation... 

Water-insoluble hormones (steroid, retinoid, and thyroid hormones) readily pass through the plasma membrane of their target cells to reach their receptor proteins in the nucleus (Fig. 23-4). With this class of hormones, the hormone-receptor complex itself carries the message it interacts with DNA to alter the expression of specific genes, changing the enzyme complement of the cell and thereby changing cellular metabolism (see Fig. 12 10). 

Smyth, M. and FitzGerald, R.J. 1998. Relationship between some characteristics of WPC hydrolysates and the enzyme complement in commercially available proteinase preparations. Int. Dairy J. 8, 819-827. 

Because the intermediary metabolism of various organs is virtually the same, organ-specific enzymes are very rare. One example usually cited is the acid phosphatase of the prostate. However, the enzyme complement of the various organs may differ with respect to relative activities of the enzymes, the time dependence of their appearance in plasma, and the pattern of their isoenzymes (see below). Table 5.2 presents a list of enzymes commonly used for organ- and disease-specific diagnoses. 

A series of proteins collectively called the complement participate in the immune response to the entry of foreign cellular or viral material into the organism. This group of proteins consists of about 20 entities, some of which are enzymes. Complement was first associated with the lysis of foreign red blood cells in the nineteenth century it also participates in the lysis of bacterial cells. The complement activation cascade, very similar to the blood coagulation cascade, involves the stepwise activation, via proteolysis, of various components of the complement system until a final protein complex, the membrane attack unit (also called the C5b-9 complex), is generated. It then punches holes in the membrane to which it is bound. 

One of the areas of assay development that has seen increased application is the use of enzyme complementation assays. While this has primarily been used in cell-based assays, a number of interesting applications of enzyme complementation for in vitro assays have also been reported.26... 

Since the initial paper by Fields and Song, there have been significant technical improvements in the method. DNA-binding domains and transcription activation domains have been optimized to reduce false positives and increase the transcription read-out. A variety of reporter plasmids have been engineered to detect a broad range of protein-protein interactions. Much more is understood about the nature of false positives and how to rout them out. Moreover, in response to the utility of this approach, several laboratories have begun to develop transcription-based assays that can be carried out in bacteria, or protein-protein interaction assays based on alternate readouts such as enzyme complementation or fluorescence resonance energy transfer (FRET). 

Rossi F, Charlton CA, Blau HM. Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc. Natl. Acad. Sci. U.S.A. 1997 94 8405-8410. Wehrman TS, Casipit CL, Gewertz NM, Blau HM. Enzymatic detection of protein translocation. Nat. Methods 2005 2 521-527. Hammer MM, Wehrman TS, Blau HM. A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization. FASEB J. 2007 21 3827-3834. 

Normally a fraction of the cellular enzyme complement is sufficient to maintain proper function and the regulation of activity is a function of substrate level. In the case of those subjects with partial deficiency of HPRT, it is possible to demonstrate the superiority of the intact cell assay as a measure of the true in vivo situation. In addition, these studies demonstrate the advantage of using an intact cell over the crude lysate in looking for an index of actual enzyme performance in tissues in general. 

DNA polymerases perform essential roles in the replication and repair of genetic material. The DNA polymerases of several mesophiles have been extensively characterized, and the structural, biochemical, and kinetic properties of Escherichia coli, yeast, mammalian, and bacteriophage DNA polymerases have been the subject of numerous reviews. " Certain enzymes complement DNA polymerase activity with 3 - -5 -exonuclease (proofreading) activity and/or 5 - 3 -exonuclease activity, which typically reside in separate structural domains on the same polypeptide. To orchestrate DNA replication and repair, both eubacteria and eukaryotes possess multiple DNA polymerases, each with distinct properties, subunit compositions, and physiological roles. In vivo, DNA polymerases require the assistance of numerous accessory proteins to replicate and repair the genome (reviewed ). Based on amino acid sequence homology to E. coli enzymes, DNA polymerases have been classified into at least three distinct families Family A (Pol I-like), Family B (Pol Il-like), and Family C (Pol Ill-like). ... 

Finally, the rate of degradation can also depend on the activity of the degrading system. The presence or absence of lysosomes and their enzyme complements, for example, may influence the degree of degradation (De Duve, 1963 Weber, 1963). 

Why use whole cells themselves rather than the relevant enzyme complement ... 

The enzyme complements for growing cultures can be divided into two... 

The active enzyme complement in most spore cultures is singularly unresponsive to changes in pH and temperature. This probably is a reflection of the fact that spores were evolved to be thick-walled cell forms, highly resistant to considerable extremes of environmental circumstances. 

An organic compound is amenable to biotransformation only if it is accessible to the relevant enzyme complement of a whole-cell biocatalyst. This presents little or no problem for those biotransformations catalysed by enzymes secreted outside the boundary wall by the cells of microorganisms. However, for the majority of whole-cell biotransformations, the relevant enzymes are retained within the cells of the micro-organisms. 

As the cotyledonary reserves are consumed the mitochondria become disorganized and gradually lose their respiratory efficiency, enzyme complement and activity. In cotyledons of dark-grown Alaska peas this is marked by a decline in respiratory control (Fig. 5.7A), a loss of efficiency of oxidative phosphorylation (shown by the fall in ADP/0 ratio) (Fig. 5.7 B) and in cytochrome oxidase activity (Fig. 5.7 C). Another mitochondrial enzyme, malate dehydrogenase does not decline however. The gradual loss of mitochondrial activity is accompanied by the disruption of cell structure (see Chap. 6). 

It is reasonable that the process of vesiculation occurs at an equilibrium rate, constant for each cell species and modified by the metabolic demands of the cells. We have suggested (Verity and Brown, 1968b), from maturation studies of cerebral lysosomal enzymes, that for a given rate of vesiculation, the enzyme complement of the primary lysosome is directly proportional to the individual rates of synthesis of the component enzymes. Such a hypothesis would account for the heterogeneity in acid hydrolase reaction to injury, the difference in maturation profiles of individual acid hydrolases, and the dysynchrony of acid hydrolase induction. Also suggested is a direct feedback mechanism whereby a stimulation of vesiculation may in turn induce increased enzyme synthesis, possibly through changes in membrane phospholipid metabolism. 

Only a few ducklings are able to survive at the low level of 50 mg/100 gm diet of tryptophan, whereas all survive at a supplement of 1 gm/100 gm diet. Although the results are still preliminary, they suggest that there may be a genetic factor in the ability of animals to utilize tryptophan as a growth factor in the absence of niacin. It is possible that the difference in response of the individual birds may be due to a quantitative or qualitative difference in the enzymes involved in the formation of niacin from tryptophan. The individuals, growing in the absence of either added nicotinamide or tryptophan, may have an enzyme complement particularly efficient in promoting the synthesis of the vitamin from tryptophan. 

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