Ectonucleotidases in Blood Malignancies: A Tale of Surface Markers and Therapeutic Targets.

Leukemia develops as the result of intrinsic features of the transformed cell, such as gene mutations and derived oncogenic signaling, and extrinsic factors, such as a tumor-friendly, immunosuppressed microenvironment, predominantly in the lymph nodes and the bone marrow. There, high extracellular levels of nucleotides, mainly NAD+ and ATP, are catabolized by different ectonucleotidases, which can be divided in two families according to substrate specificity: on one side those that metabolize NAD+, including CD38, CD157, and CD203a; on the other, those that convert ATP, namely CD39 (and other ENTPDases) and CD73. They generate products that modulate intracellular calcium levels and that activate purinergic receptors. They can also converge on adenosine generation with profound effects, both on leukemic cells, enhancing chemoresistance and homing, and on non-malignant immune cells, polarizing them toward tolerance. This review will first provide an overview of ectonucleotidases expression within the immune system, in physiological and pathological conditions. We will then focus on different hematological malignancies, discussing their role as disease markers and possibly pathogenic agents. Lastly, we will describe current efforts aimed at therapeutic targeting of this family of enzymes.

3'-nucleotidase/nuclease activity allows Leishmania parasites to escape killing by neutrophil extracellular traps.

Leishmaniasis is a widespread neglected tropical disease caused by parasites of the Leishmania genus. These parasites express the enzyme 3'-nucleotidase/nuclease (3'NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3'NT/NU in Leishmania survival of NET-mediated killing. Promastigotes of Leishmania infantum were cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups of Leishmania during interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3'NT/NU inhibitor. Inhibition of 3'NT/NU by 3'-AMP, 5'-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show that Leishmania infantum induces NET release and that promastigotes can escape NET-mediated killing by 3'-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

Neuromodulation: purinergic signaling in respiratory control.

The main functions of the respiratory neural network are to produce a coordinated, efficient, rhythmic motor behavior and maintain homeostatic control over blood oxygen and CO2/pH levels. Purinergic (ATP) signaling features prominently in these homeostatic reflexes. The signaling actions of ATP are produced through its binding to a diversity of ionotropic P2X and metabotropic P2Y receptors. However, its net effect on neuronal and network excitability is determined by the interaction between the three limbs of a complex system comprising the signaling actions of ATP at P2Rs, the distribution of multiple ectonucleotidases that differentially metabolize ATP into ADP, AMP, and adenosine (ADO), and the signaling actions of ATP metabolites, especially ADP at P2YRs and ADO at P1Rs. Understanding the significance of purinergic signaling is further complicated by the fact that neurons, glia, and the vasculature differentially express P2 and P1Rs, and that both neurons and glia release ATP. This article reviews at cellular, synaptic, and network levels, current understanding and emerging concepts about the diverse roles played by this three-part signaling system in: mediating the chemosensitivity of respiratory networks to hypoxia and CO2/pH; modulating the activity of rhythm generating networks and inspiratory motoneurons, and; controlling blood flow through the cerebral vasculature.

Cellular function and molecular structure of ecto-nucleotidases.

Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ecto-nucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

Some aspects of purinergic signaling in the ventricular system of porcine brain.

BACKGROUND: Numerous signaling pathways function in the brain ventricular system, including the most important - GABAergic, glutaminergic and dopaminergic signaling. Purinergic signalization system - comprising nucleotide receptors, nucleotidases, ATP and adenosine and their degradation products - are also present in the brain. However, the precise role of nucleotide signalling pathway in the ventricular system has been not elucidated so far. The aim of our research was the identification of all three elements of purinergic signaling pathway in the porcine brain ventricular system. RESULTS: Besides nucleotide receptors on the ependymocytes surface, we studied purines and pyrimidines in the CSF, including mechanisms of nucleotide signaling in the swine model (Sus scrofa domestica). The results indicate presence of G proteins coupled P2Y receptors on ependymocytes and also P2X receptors engaged in fast signal transmission. Additionally we found in CSF nucleotides and adenosine in the concentration sufficient to P receptors activation. These extracellular nucleotides are metabolised by adenylate kinase and nucleotidases from at least two families: NTPDases and NPPases. A low activity of these nucleotide metabolising enzymes maintains nucleotides concentration in ventricular system in micromolar range. ATP is degraded into adenosine and inosine. CONCLUSIONS: Our results confirm the thesis about cross-talking between brain and ventricular system functioning in physiological as well as pathological conditions. The close interaction of brain and ventricular system may elicit changes in qualitative and quantitative composition of purines and pyrimidines in CSF. These changes can be dependent on the physiological state of brain, including pathological processes in CNS.

The combined effect of environmental and host factors on the emergence of viral RNA recombinants.

Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.

Regulation by degradation, a cellular defense against deoxyribonucleotide pool imbalances.

Deoxyribonucleoside triphosphates (dNTPs) are the precursors used by DNA polymerases for replication and repair of nuclear and mitochondrial DNA in animal cells. Accurate DNA synthesis requires adequate amounts of each dNTP and appropriately balanced dNTP pools. Total cellular pool sizes are in the range of 10-100pmoles of each dNTP/million cells during S phase, with mitochondrial pools representing at most 10% of the total. In quiescent or differentiated cells pools are about 10-fold lower both in the cytosol and mitochondria. Contrary to what may be expected on the basis of the roughly equimolar abundance of the 4 nitrogen bases in DNA, the four dNTPs are present in the pools in different ratios, with pyrimidines often exceeding purines. Individual cell lines may exhibit different pool compositions even if they are derived from the same animal species. It has been known for several decades that imbalance of dNTP pools has mutagenic and cytotoxic effects, and leads to "mutator" phenotypes characterized by increased mutation frequencies. Until 10 years ago this phenomenon was considered to affect exclusively the nuclear genome. With the discovery that thymidine phosphorylase deficiency causes destabilization of mitochondrial DNA and a severe multisystemic syndrome the importance of dNTP pool balance was extended to mitochondria. Following that first discovery, mutations in other genes coding for mitochondrial or cytosolic enzymes of dNTP metabolism have been associated with mitochondrial DNA depletion syndromes. Both excess and deficiency of one dNTP may be detrimental. We study the mechanisms that in mammalian cells keep the dNTP pools in balance, and are particularly interested in the enzymes that, similar to thymidine phosphorylase, contribute to pool regulation by degrading dNTP precursors. The role of some relevant enzymes is illustrated with data obtained by chemical or genetic manipulation of their expression in cultured mammalian cells.

[Ecto-nucleotidases of ectonucleoside triphosphate diphosphohydrolase family: structure, localization and functional significance].

Ecto-nucleotidases are enzymes of hydrolase class. They split extracellular nucleoside tri- and diphosphate. In this review a short history of these enzymes investigation, classification, structure, and functional significance of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase) has been presented. These enzymes are glycoproteins anchored in membranes. They do not form phosphorylated enzyme's form during catalytic circle, and (by analogy with membrane-bound ATPases) form homooligomeric ensembles. Activity of these enzymes depends on bivalent ions, in particular Ca2+ and Mg2+. E-NTPDases function in the composition of ecto-nucleotidase cascade that contains other nucleotide-hydrolyzing enzymes. They regulate P2-receptors by hydrolyzing its ligand specifically ATP. Both modern information and results of our investigation about influence of different endo- and exogenous factors on activity of these enzymes has been presented.

[Purinergic signals]. / Señales purinérgicas.

In the last decade evidence accumulated that nucleosides and nucleotides of both uridine and adenine can act as extracellular signaling factors. Their action is mediated by two main types of surface receptors commonly known as purinergic. P1 receptors are metabotropic and activated by adenosine, whereas receptors for nucleotides (ATP, ADP, UTP and UDP) and nucleotide-sugars (UDP-glucose and UDP-galactose) can be either metabotropic (P2Y) or ionotropic (P2X). The importance and complexity of this signaling system is evidenced by various mechanisms of nucleotide release, as well as by the ibiquitous distribution of various types of ectonucleotidases which catalyze and convert extracellular nucleotides. Up to now about twenty receptors have been cloned and found to modulate the nerve impulse, inflammatory response, insuline secretion, the regulation of the vascular tone and nociception, among other processes. In the present review we describe the main structural and pharmacological features of purinergic receptors, and analyze how the dynamic interaction between these receptors, nucleotides and nucleosides, and ectonucleotidases modulate several biological responses. Particular focus is given to platelet aggregation and thrombus formation, the immune response and the hydration of the mucosal linings of the respiratory tract.

Señales purinérgicas / Purinergic signals

En la última década se ha aportado clara evidencia de que tanto nucleósidos como nucleótidos de adenina y uridina pueden funcionar como factores de señalización extracelular. Su acción es mediada por dos tipos principales de receptores de superficie denominados purinérgicos. Los receptores P1 se activan por adenosina, y son todos metabotrópicos, mientras que los receptores de nucleótidos (ATP, ADP, UTP y UDP) y nucleótidos-azúcares (UDP-glucosa y UDP-galactosa) pueden ser metabotrópicos (P2Y) o ionotrópicos (P2X). La importancia y complejidad de este sistema de señalización se evidencia por la diversidad de mecanismos de liberación de nucleótidos al medio extracelular y por la distribución ubicua de varios grupos de ectonucleotidasas capaces de catalizar la degradación y conversión de nucleótidos. Hasta el momento se han descrito y clonado una veintena de estos receptores que modulan una variedad de respuestas, como el impulso nervioso, la respuesta inflamatoria, la secreción de insulina, la regulación del tono vascular y la percepción del dolor. En la presente revisión se describen las características estructurales y farmacológicas de los receptores purinérgicos y se analiza la interacción dinámica entre estos receptores, los nucleósidos y nucleótidos, y las ectonucleotidasas, con especial atención a la dinámica de la agregación plaquetaria, la respuesta inmune y la hidratación de las mucosas respiratorias.

In the last decade evidence accumulated that nucleosides and nucleotides of both uridine and adenine can act as extracellular signaling factors. Their action is mediated by two main types of surface receptors commonly known as purinergic. P1 receptors are metabotropic and activated by adenosine, whereas receptors for nucleotides (ATP, ADP, UTP and UDP) and nucleotide-sugars (UDP-glucose and UDP-galactose) can be either metabotropic (P2Y) or ionotropic (P2X). The importance and complexity of this signaling system is evidenced by various mechanisms of nucleotide release, as well as by the ibiquitous distribution of various types of ectonucleotidases which catalyze and convert extracellular nucleotides. Up to now about twenty receptors have been cloned and found to modulate the nerve impulse, inflammatory response, insuline secretion, the regulation of the vascular tone and nociception, among other processes. In the present review we describe the main structural and pharmacological features of purinergic receptors, and analyze how the dynamic interaction between these receptors, nucleotides and nucleosides, and ectonucleotidases modulate several biological responses. Particular focus is given to platelet aggregation and thrombus formation, the immune response and the hydration of the mucosal linings of the respiratory tract.

Rv2131c from Mycobacterium tuberculosis is a CysQ 3'-phosphoadenosine-5'-phosphatase.

Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3'-phosphoadenosine-5'-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the Mtb genome suggests the presence of a CysQ homologue encoded by the gene Rv2131c. However, a recent biochemical study assigned the protein's function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed Rv2131c heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl 2. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a K m (8.1 +/- 3.1 microM) and k cat (5.4 +/- 1.1 s (-1)) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the Rv2131c-encoded enzyme to dephosphorylate PAP and PAPS in vivo was confirmed by functional complementation of an Escherichia coli Delta cysQ mutant. Taken together, these studies indicate that Rv2131c encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism.

Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5'-3' exonucleases Rat1 and Xrn1.

Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD) pathway, since mature tRNA(Val(AAC)) lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and deacylated at 37 degrees C in a trm8-Delta trm4-Delta strain, resulting in temperature-sensitive growth. We show here that components of this RTD pathway include the 5'-3' exonucleases Rat1 and Xrn1, and Met22, which likely acts indirectly through Rat1 and Xrn1. Since deletion of MET22 or mutation of RAT1 and XRN1 prevent both degradation and deacylation of mature tRNA(Val(AAC)) in a trm8-Delta trm4-Delta strain and result in healthy growth at 37 degrees C, hypomodified tRNA(Val(AAC)) is at least partially functional and structurally intact under these conditions. The integrity of multiple mature tRNA species is subject to surveillance by the RTD pathway, since mutations in this pathway also prevent degradation of at least three other mature tRNAs lacking other combinations of modifications. The RTD pathway is the first to be implicated in the turnover of mature RNA species from the class of stable RNAs. These results and the results of others demonstrate that tRNA, like mRNA, is subject to multiple quality control steps.

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression.

Previous studies indicated that the vaccinia virus D10 protein, which is conserved in all sequenced poxviruses, participates in the rapid turnover of host and viral mRNAs. D10 contains a motif present in the family of Nudix/MutT enzymes, a subset of which has been shown to enhance mRNA turnover in eukaryotic cells through cleavage of the 5' cap (m7GpppNm-). Here, we demonstrate that a purified recombinant D10 fusion protein possesses an intrinsic activity that liberates m7GDP from capped RNA substrates. Furthermore, point mutations in the Nudix/MutT motif abolished decapping activity. D10 has a strong affinity for capped RNA substrates (Km approximately 3 nm). RNAs of 24-309 nt were decapped to comparable extents, whereas the cap of a 12-nt RNA was uncleaved. At large molar ratios relative to capped RNA substrate, competitor m7GpppG, m7GTP, or m7GDP inhibited decapping, whereas even higher concentrations of unmethylated analogs did not. High concentrations of uncapped RNA were also inhibitory, suggesting that D10 recognizes its substrate through interaction with both cap and RNA moieties. Thus far, poxviruses represent the only virus family shown to encode a Nudix hydrolase-decapping enzyme. Although it may seem self-destructive for a virus to encode a decapping and a capping enzyme, accelerated mRNA turnover helps eliminate competing host mRNAs and allows stage-specific synthesis of viral proteins.

Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans.

Extracellular ATP and ADP trigger vasodilatatory and prothrombotic signalling events in the vasculature. Here, we tested the hypothesis that nucleotide turnover is activated in the bloodstream of exercising humans thus contributing to the enhanced platelet reactivity and haemostasis. Right atrial, arterial and venous blood samples were collected from endurance-trained athletes at rest, during submaximal and maximal cycle ergometer exercise, and after early recovery. ATP-specific bioluminescent assay, together with high-performance liquid chromatographic analysis, revealed that plasma ATP and ADP concentrations increased up to 2.5-fold during maximal exercise. Subsequent flow cytometric analysis showed that plasma from exercising subjects significantly up-regulated the surface expression of P-selectin in human platelets and these prothrombotic effects were diminished after scavenging plasma nucleotides with exogenous apyrase. Next, using thin layer chromatographic assays with [gamma-(32)P]ATP and (3)H/(14)C-labelled nucleotides, we showed that two soluble nucleotide-inactivating enzymes, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase, constitutively circulate in human bloodstream. Strikingly, serum nucleotide pyrophosphatase and hydrolase activities rose during maximal exercise by 20-25 and 80-100%, respectively, and then declined after 30 min recovery. Likewise, soluble nucleotidases were transiently up-regulated in the venous blood of sedentary subjects during exhaustive exercise. Human serum also contains 5'-nucleotidase, adenylate kinase and nucleoside diphosphate (NDP) kinase; however, these activities remain unchanged during exercise. In conclusion, intravascular ADP significantly augments platelet activity during strenuous exercise and these prothrombotic responses are counteracted by concurrent release of soluble nucleotide-inactivating enzymes. These findings provide a novel insight into the mechanisms underlying the enhanced risk of occlusive thrombus formation under exercising conditions.

Kinetics of urothelial ATP release.

Recent reports have proposed that the urothelium can sense mechanical stretch and communicate this information to sensory afferent neurons by the release of ATP into the vicinity of P2X-containing neurons. This report investigates the bidirectional release of ATP by in vitro rabbit urothelium. ATP was measured using the luciferin-luciferase assay. Immediately after washing of both sides of the epithelium, there was a linear increase in ATP content in the mucosal compartment with a rate of 23 +/- 6.5 fmol x min(-1) x cm(-2) (n = 18). Serosal ATP content increased as a saturating exponential function, suggesting a constant rate of release and degradation of ATP by ectonucleotidases/exonucleotidases. The presence of a serosal ectonucleotidase/exonucleotidases was demonstrated by the time-dependent decrease in exogenously added ATP. The maximum rate of hydrolysis was 11 pmol x min(-1) x cm(-2) with a K(m) of 0.49 microM. The time course of serosal ATP release was modeled as a constant rate of release (d: mol x min(-1) x cm(-2)) and rate constant of hydrolysis (k(h): min(-)). In control conditions d was 18 fmol x min(-1) x cm(-2) and k(h) of 0.056 +/- 0.01 min(-) (n = 18). Steady-state serosal chamber content is 370 +/- 90 fmol/cm(2), and concentration is 50 +/- 1.2 x 10(-12) M. Stretching the tissue resulted in a transient fivefold increase in the rate of mucosal ATP release and a transient sixfold increase in serosal ATP release. Half-osmotic strength solutions increased mucosal release by 10-fold and serosal release by 5-fold. Tissue damage resulted in a step-increase in mucosal chamber ATP content by 6.6 +/- 1 pmol/cm(2) and serosal chamber ATP by 0.1 +/- 0.06 pmol/cm(2) (n = 5).

Structure and activity analyses of Escherichia coli K-12 NagD provide insight into the evolution of biochemical function in the haloalkanoic acid dehalogenase superfamily.

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 A with R(work) = 19.8% and R(free) = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with k(cat)/K(m) = 3.12 x 10(4) and 1.28 x 10(4) microM(-)(1) s(-)(1) for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k(cat)/K(m)) are low (1 x 10(4) M(-)(1) s(-)(1)) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.

The 5'-nucleotidases as regulators of nucleotide and drug metabolism.

The 5'-nucleotidases are a family of enzymes that catalyze the dephosphorylation of nucleoside monophosphates and regulate cellular nucleotide and nucleoside levels. While the nucleoside kinases responsible for the initial phosphorylation of salvaged nucleosides have been well studied, many of the catabolic nucleotidases have only recently been cloned and characterized. Aside from maintaining balanced ribo- and deoxyribonucleotide pools, substrate cycles that are formed with kinase and nucleotidase activities are also likely to regulate the activation of nucleoside analogues, a class of anticancer and antiviral agents that rely on the nucleoside kinases for phosphorylation to their active forms. Both clinical and in vitro studies suggest that an increase in nucleotidase activity can inhibit nucleoside analogue activation and result in drug resistance. The physiological role of the 5'-nucleotidases will be covered in this review, as will the evidence that these enzymes can mediate resistance to nucleoside analogues.

Serum enzymes associated with cholestasis.

Alkaline phosphatase, gamma-glutamyl transferase, and 5' nucleotidase are the most common enzymes used in the evaluation of cholestasis. The present knowledge of these enzymes including their function, activity measurement, biologic variables of enzyme activity in healthy persons and disease states, and clinical significance are reviewed. Usefulness of enzymes patterns for diagnosis of specific cholestatic disorders and future directions in evaluation of cholestasis are also discussed.