**1. Introduction**

In this chapter, we intend to detail basic protocols for the in situ detection of ecto-nucleotidases as an introduction to the technique for those who have never made these experimental approaches. This chapter does not aim to be a review on ecto-nucleotidases because there are already excellent highly recommended reviews [1–4].

Ecto-nucleotidases are broadly expressed enzymes active in almost all tissues of all organisms, both animals and plants. What varies among the cell (and tissue) types are the subtype(s) of enzyme(s) and the combination of them, expressed in a particular cell type. In general, these enzymes convert adenosine triphosphate (ATP), as well as diphosphate (ADP) and monophosphate (AMP), into adenosine. In situ detection of these enzymes confers functional sense on immunodetection studies. It is also a convenient tool for the validation of new inhibitors of these enzymes, which can be studied in the cell context of the tissue where they are found. The study of ecto-nucleotidases and their inhibitors (many of them antibodies) is at the center of oncological research to therapeutically target the adenosinergic pathway, a fact reflected in the increased number of high impact publications in the field.

The technique is feasible because ecto-nucleotidases maintain their activity of hydrolyzing nucleotides in formalin-fixed frozen tissues (and cells). Inorganic phosphorous (Pi) generated upon their activity combines with a lead salt added to the reaction mixture, forming brown precipitates in the places where the enzymes are active, which can be visualized under light microscope. The protocol, with slight modifications, can also be used for electron microscopy.

There are four families of membrane-bound ecto-nucleotidases. Other nucleotidases act intracellularly but are not studied here. The main features of ectonucleotidases are included in **Figure 1** and summarized below.

#### **1.1 Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases)**

The E-NTPDase family is composed of eight members, four of which are cell surface-located: NTPDase1, also known as CD39; NTPDase2 or CD39L1; NTPDase3 or CD39L3; and NTPDase8. They perform the ATP (and ADP) hydrolysis to AMP with different ADP production abilities. These differences between enzymes reflect different consequences in cells depending on the ATP receptors expressed [5]. The four members display similar structural properties, with two transmembrane domains, close to the N and C terminus, and a catalytic extracellular domain [3]. They require millimolar concentrations of Mg2+ and Ca2+ ions in order to perform ATP hydrolysis, and the absence of these ions results in no enzymatic activity. All of them hydrolyze nucleoside triphosphates (NTP), but they differ in substrate specificity. NTPDase1 hydrolyzes ATP and ADP equally, while NTPDase3 and NTPDase8 hydrolyze ATP or uridine triphosphate (UTP) more efficiently than ADP or uridine diphosphate (UDP). Finally, the NTPDase2 is the most ATP-specific NTPDase, and for this reason it is also named the ecto-ATPase [2].

Most of the available NTPDase inhibitors are ATP analogues such as ARL-67156 and PSB-6426, a potent NTPDase2 inhibitor. Non-nucleotide-based inhibitors also described in literature are compounds related to dyes bearing sulfonate groups such

#### **Figure 1.**

*Schematic representation of the four families of membrane-bound ecto-nucleotidases and their substrate specificities. E-NTPDases, ecto-nucleoside triphosphate diphosphohydrolases; E-NPPs, ecto-nucleotide pyrophosphatase/phosphodiesterases; ecto-5'-NT, ecto-5'-nucleotidase; APs, alkaline phosphatases; NTP, nucleoside triphosphate; NDP, nucleoside diphosphate; NMP, nucleoside monophosphate; cNMP, cyclic nucleoside monophosphate; N, nucleoside.*

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*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides*

as suramin, a nonselective inhibitor, and the pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Other inhibitors are the polyoxometalates, such as POM 1 [6]. Inhibitory antibodies, mainly against CD39, are being developed for use

The E-NPP family represents a versatile group of seven structurally related enzymes with pyrophosphatase and phosphodiesterase activities having a wide range of hydrolysable substrates. The membrane-bound ectoenzymes NPP1 and NPP3 and the secreted NPP2 are the most studied members. Catalytic activity of E-NPPs is composed of a two-step hydrolysis consisting of a first attack on the phosphate of the incoming substrate by a threonine/serine metal-activated catalytic site and a second attack on the intermediate substrate by a metalactivated site, thus releasing a nucleoside 5′-monophosphate. In general, NPP1–3 are typed as alkaline ecto-nucleotide pyrophosphatases that hydrolyze a number of phosphodiester bonds (e.g., from oligonucleotides, lysophosphatidylcholine, sphingomyelin, and glycerophosphorylcholine or from artificial substrates like the p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP)) and pyrophosphate bonds (e.g., from (d)NTPs, (d)NDPs, NAD, FAD, and UDP sugars or from artificial substrates like the thiamine pyrophosphate (TPP)) to generate nucleoside 5′-monophosphates. TPP is the "false" substrate mainly used for NPP identification in in situ activity assays. Like most of the enzymes, E-NPPs can be inhibited in vitro by the substrates and products of the NPP reaction, as well as by heparin and heparan sulfate glycosaminoglycans, and by other substances such as imidazole, 2-mercaptoethanol, and metal ion-chelating agents [8]. Anti-NPP3 inhibitory antibody represents a promising therapeutic tool for the treatment of renal cell

Extracellular AMP resulting from the hydrolysis of ATP and ADP by most of the ecto-nucleotidases can in turn be efficiently hydrolyzed into adenosine by eN, a glycosylphosphatidylinositol-linked membrane-bound glycoprotein also known as CD73 [10], which hydrolyses nucleotide-5′-monophosphates (NMP) [3]. It is broadly expressed as an alpha dimer bound with disulfide bridges and shows different functions depending on the cell type. Although eN activity is ion-independent in physiological conditions, in vitro the presence of Mg2+ ions can considerably increase its ability to hydrolyze AMP. In addition to its AMPase activity, eN hydrolyzes 2-deoxyribose compounds but much less effectively than AMP. Unlike other ectoenzymes such as NPPs, eN is not inhibited by Pi. Alpha,beta-methylene-ADP and some of its derivatives and analogues are efficient inhibitors [11]. Inhibitory anti-

Phosphatases are a superfamily of proteins that mediate the phosphate removal of proteins and other substrates [12]. Depending on their substrate specificity, they are divided into two major groups: the protein phosphatases, which mediate the hydrolysis of phosphate groups from protein residues (e.g., serine/threonine phosphatases), and the membrane-bound phosphatases, which mediate the hydrolysis of phosphate groups from nonprotein substrates (e.g., acid and alkaline phosphatases). In this chapter, we are focusing on the membrane-bound phosphatases, in

**1.2 Ecto-nucleotide pyrophosphatases/phosphodiesterases (E-NPPs)**

*DOI: http://dx.doi.org/10.5772/intechopen.84495*

in cancer therapy [7].

carcinoma [9].

**1.3 Ecto-5′-nucleotidase (ecto-5′-NT, eN)/CD73**

CD73 antibodies are used in clinical trials [7].

**1.4 Alkaline phosphatases (APs)**

particular the AP family [12, 13].

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides DOI: http://dx.doi.org/10.5772/intechopen.84495*

as suramin, a nonselective inhibitor, and the pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Other inhibitors are the polyoxometalates, such as POM 1 [6]. Inhibitory antibodies, mainly against CD39, are being developed for use in cancer therapy [7].
