Adenosine deaminase assay

Identification of proteins that bind to Z-DNA added one further step to the establishment of the presence of Z-DNA in vivo and its possible biological role. Herbert and Rich [22] demonstrated an in vitro assay system where one type of double-stranded RNA adenosine deaminase, called DRAD-binding Z-DNA. There are evidences that topoisomerase II from Drosophila, hiunan and calf thymus recognizes a number of DNA shapes, including Z-DNA [34,35]. Bloomfield and coworkers [36] have found that the condensation of plasmids is enhanced by Z-DNA conformation in d(CG)n repeats. The information related to B-Z transition [31], the effect of ligands on it [28,29] and X-ray crystal structure data [37,38] appear to suggest that the possible biological role of this polymorphic form of DNA will be soon established. 

The activity of hypoxanthine-guanine phosphoribosyltransferase, adenine phos-phoribosyltransferase, adenosine deaminase, and purine nucleoside phosphorylase can be determined in dried blood spots using an HPLC-linked assay [3]. 

ADA BEVS BHK CHO ELISA mAb MDCK MMR SCID tPA VLP adenosine deaminase deficiency baculovirus expression vector system baby hamster kidney cell line Chinese hamster ovary cell line enzyme linked immuno sorbent assay monoclonal antibodies Madin-Darby canine kidney epithelial cells measles, mumps, rubella severe combined immunodeficiency plasminogen activator virus-like particle... 

Adenosine deaminase catalyzes the deamination of adenosine to form inosine and ammonia. The inosine (Ino) can be degraded further to hypoxanthine (Hyp) by nucleoside phosphorylase, an activity often present in extracts. Therefore, in many cases, the assay involves a determination of either the loss of adenosine (Ado) or the formation of both inosine and hypoxanthine. An early study by Uberti et al., 1977, was followed by another by Hartwick et al., 1978. 

Pennington (PI) and Uberti (U1) first introduced the technique of liquid chromatographic enzyme assays by using the ion-exchange mode of HPLC in their analyses of 3, 5 -cyclic adenosine monophosphate phosphodiesterase and adenosine deaminase, respectively. Since that time, a number of liquid chromatographic enzyme assays have been developed. 

Hartwick et al. (Hll) used RPLC in developing an assay for adenosine deaminase which has been optimized for pH, ionic strength, reaction time, and substrate concentration. With this technique the substrate adenosine was monitored simultaneously with the products, hypoxanthine and in-osine. 

The salvage pathway does not involve the formation of new heterocyclic bases but permits variation according to demand of the state of the base (B), i.e. whether at the nucleoside (N), or nucleoside mono- (NMP), di- (NDP) or tri- (NTP) phosphate level. The major enzymes and routes available (Scheme 158) all operate with either ribose or 2-deoxyribose derivatives except for the phosphoribosyl transferases. Several enzymes involved in the biosynthesis of purine nucleotides or in interconversion reactions, e.g. adenosine deaminase, have been assayed using a method which is based on the formation of hydrogen peroxide with xanthine oxidase as a coupling enzyme (81CPB426). 

An assay for adenosine involves the primary enzyme adenosine deaminase, and a chemical indicator reaction that consumes ammonia by reaction with the ninhy-drin reagent (Eq. 3.4) ... 

S2, S8). Others have reported that adenosine inhibits adenylate cyclase from brain, adipocytes, and lung tissue (FI, M6, Wl). Brain tissues of most animals have very little adenosine deaminase. In other tissues the role of breakdown products must be further explored. The fact that cyclase in intact slices is stimulated while that of broken-cell preparations is inhibited may be related to the so-called latent adenosine deaminase that is mitochondria-bound and inactive in assays of adenosine deaminase in homogenates (M16). 

Assay of adenosine deaminase and of other enzymes of purine and pyrimidine metabolism were performed by previously described radioisotopic assays (3). Electrophoresis of RBC ADA was performed on hemolysates after Spencer et al. (4). The enzymes of qlu-cose and glutathione metabolism were assayed after Beutler (5). Erythrocyte glucose consumption was assayed according to Cartier et al. (6). Adenosine deaminase was partially purified from human red blood cells the specific activity of the preparation was 1.2 pmol.min l.mg protein l. Rabbit antiserum to human adenosine deaminase was raised by immunization of white male rabbits with this preparation. 

TL Kjellstrom, L Bachas. Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label. Anal Chem 61 1728-1732, 1989. 

The enzymatic reactions leading to MTA from Ado-Met (see Fig. 3) can also be assayed spectrophotometrically by using adenosine deaminase (EC 3.5.4.4), which is very effective in deaminating MTA,... 

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