Enzyme activities, microarrays

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

Sieber SA, Mondala TS, Head SR et al (2004) Microarray platform for profiling enzyme activities in complex proteomes. J Am Chem Soc 126 15640-15641... 

Besides enzyme activity, enzyme induction assays can also utilize mRNA and enzyme protein level as endpoints. Gene expression studies now can be performed using branch-chained DNA and microarray techniques. Protein level quantification in general is performed using isoform-specific antibodies and Western blotting. Enzyme activity represents the most relevant endpoint for drug-drug interaction evalua-... 

Small molecule or peptide substrates can also be used to profile protease and other enzyme activities. Salisbury et al. described a protease-substrate microarray in which the carboxyl end of the peptide substrates was conjugated to 7-amino-4-car-bamoylmethyl coumarin, a fluorogenic compound. The conjugate was non-fluores-cent when the electron-donating group on the coumarin was attached to the peptide. Upon proteolysis, the peptide was released and the microarray spot fluoresced. Zhu et al. demonstrated that small molecule microarrays could be used to detect enzyme activities of epoxide hydrolases and phosphatases. 

Analysis of metabohc networks may help us properly interpret enzyme activities from enzyme abundance, and ultimately extrapolate to the whole organism using models. Apphcation of such computational modeling to enzyme systems is still limited but promising. For example, Su et al. (2006) were able to develop computational models of N assimilation in Synechococcus sp. WH 8102 that closely corresponded to results from microarray analyses of gene expression. 

An activity-based probe meeting these requirements could, in principle, enable the comparative measurement and molecular identification of all the active members of a given enzyme class present in one or more proteomes. Importantly, these enzyme activity profiles can be read out in a variety of formats including gels [20,25], microarrays [26], liquid chromatography-mass spectrometry (LC-MS) [27], and capillary electrophoresis [28] (Fig. 7.3-3). 

The family of HDAC enzymes has been named after their first substrate identified, i.e., the nuclear histone proteins. Histone proteins (H2A, H2B, H3 and H4) form an octamer complex, around which the DNA helix is wrapped in order to establish a condensed chromatin structure. The acetylation status of histones is in a dynamic equilibrium governed by histone acetyl transferases (HATs), which acetylate and HDACs which are responsible for the deacetylation of histone tails (Fig. 1). Inhibition of the HDAC enzyme promotes the acetylation of nucleosome histone tails, favoring a more transcriptionally competent chromatin structure, which in turn leads to altered expression of genes involved in cellular processes such as cell prohferation, apoptosis and differentiation. Inhibition of HDAC activity results in the activation of only a limited set of pre-programmed genes microarray experiments have shown that 2% of all genes are activated by structmally different HDAC inhibitors [1-5]. In recent years, a growing number of additional nonhistone HDAC substrates have been identified, which will be discussed in more detail below. 

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