Ada protein

An S4 coordination sphere of the type seen in the Ada protein, where four cysteines coordinate, was achieved with the tridentate tris(2-mercapto-l-phenylimidazolyl)hydroborato ligand. The zinc thiophenolate derivative showed reactivity of the thiolate linkage (Figure 8).506... 

Adenosine deaminase (ADA)—Protein absent in a type of inherited defect in the body s defense system. The absence of this protein results in death of key cells in the defense system. 

Fig. 1.25. Regulation of alkylation repair in E. coK by methylation of the Ada protein. The effect of methylating agents, such as N-nitroso-N-methyl urea lead to the formation of methyl phospho-triesters (P-Me) of DNA, as well as various base adducts. The Ada protein possesses an N-termi-nal and a C-terminal domain. In one of the first steps of alkylation repair the methyl groups of the phosphotriester is transferred to the Ada protein. The Ada protein is methylated on a Cys residue at its N-terminal domain and thereby transformed into an active transcription activator. In its methylated form the Ada protein binds to the control region of various genes to stimulate their transcription. Among the genes under the control of the Ada protein are its own gene, as well others required for DNA repair (alkB, alkA). After Lindahl et al., 1988.

In E. coli, repair is induced by alkylation damage. The repair of methylated DNA is performed by, among others, the Ada protein which possesses S-alkyl transferase and transcription activation activity. The Ada protein is methylated during the first repair process. In the methylated form the Ada protein can function as a transcriptional activator. It binds to the corresponding DNA element of an operon which encodes for... 

Ada and other proteins. Upon the binding of the methylated Ada protein to its cognate DNA element, the transcription of the genes is stimulated such that more repair proteins are available. 

There are other types of transcriptional activators in bacteria. One is transcription factor 1 (TF1) encoded by a Bacillus subtilis phage. It is a member of the protein HU family (Chapter 27). However, unlike the nonspecific HU it binds to some sites specifically and activates transcription.143 The E. coli Ada protein is the acceptor protein in removal of methyl groups from DNA (Chapter 27). The same protein is an inducer of transcription of DNA repair enzymes in the large ada regulon. Methylation of Cys 69 of the Ada protein itself converts it into a gene activator.144... 

A structural analog for the Cys4Zn motif of the ADA protein is constituted by the thiolate complex [ZnSPh(Tmph)].132 Related cadmium complexes have been also described.149 These studies are also pertinent to other enzymes that feature cysteine thiolate alkylation such as methionine synthase, methanohcoenzyme methyltransferase, farnesyl transferase. 

Fig. 2. The tryptophan anisotropy of the Ada protein ( ) on interaction with DNA at two protein concentrations (a) 2.0 M (b) 0.2 M. No change was observed in the steady-state fluorescence signal (O). The dashed lines are extrapolations to estimate the site size. The solid line is the theoretical curve for an association constant of 4 x lo A/" and a site size of 7 bp assuming no site-site interactions. (From Takahashi et a(. )...

The measurement of the tryptophan anisotropy has the additional advantage that it can be used to establish the polymeric state of the protein. In the case of the Ada protein, the anisotropy was independent of protein concentration, consistent with the observation that the protein remains in a monomeric state over a wide range of protein concentrations. An increase in the steady-state tryptophan anisotropy of the trp repressor on binding a 26 bp operator sequence has also been observed. 

Anbazhagan, V. Damai, R. S. Paul, A. Swamy, M. J. Interaction of the major protein from bovine seminal plasma, PDC-109 with phospholipid membranes and soluble ligands investigated by fluorescence approaches. Biochim. Biophys. Ada Proteins Proteomics, 2008, 1784(6), 891-899. 

The ligation of substrate to zinc presumably lowers the thiol pK in all of these proteins, but as noted by Lipscomb and Strater (30), would also be expected to decrease the nucleophilicity of the thiolate. Thus the enhanced reactivity that is conferred by zinc ligation remains to be fully explained. For the case of the Ada protein where zinc is coordinated by four cysteines Lipscomb and Strater (30) have proposed that charge transfer from the additional cysteinate ligands helps to maintain the nucleophilic character of the reactive thiolate. 

The quantities of ADA-cross-reacting material (CRM) in the red cell lysates of the parents and a sister of the proposita were found to be more or less proportional to the observed enzymatic activities, which were about half of the normal average (table 1). This might be an indication, that no detectable ADA protein produced by the mutant allele was present in their cells. It should, however, be noted, that red blood cells are not the most suitable material to study the natu re of possibly unstable mutant proteins, since they can not synthesize new protein. 

The RIA for ADA in the somatic cell hybrids has certain advantages Their ADA protein is freshly synthesized, qy ntification of ADA-CRM is not hampered by the presence of ADA-L" Furthermore,... 

We next determined if this increase was due to a change in the rate of synthesis or degradation of the ADA protein. Parental and dCF cells were pulse-labeled with 3H-leucine and l C-leucine, respectively. Labeled cell extracts were mixed and the ratio of 3h/ C present in total soluble protein determined. ADA protein was immunoprecipitated from mixed cell extracts with purified IgG. The immunoprecipitates were run on SDS polyacrylamide gels, the gels sliced, and slices containing ADA assayed for radioactivity. Ratios of 3h/14c for total protein synthesis and for ADA protein were compared. Table 3 shows that the relative rate of ADA synthesis clearly paralleled the relative enzyme levels in 3 resistant cell lines with different levels of ADA activity. Degradation rates of ADA, on the other hand, did not differ significantly in the sensitive or resistant cells. 

ADA mBNA activity was measured by vitro translation of (8) polysomal RNA in a rabbit reticulocyte system. The amount of ADA protein synthesized was determined by immunoprecipitation and gel electrophoresis. One cell line tested, 4-3-15, which has a 181-fold increase in ADA activity had a corresponding 171-fold increase in mRNA activity as compared to the dCF-sensitive parental cell line. 

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