Optimization of the method for determination of the catalytic activity of ecto-ATPase in human serum

Ekto-ATPaze su transmembranski enzimi koji sudjeluju u defosforilaciji izvanstanicnog ATP-a i ADP-a. Izvanstanicni ATP te drugi nukleotidi i nukleozidi su važne signalne molekule koje reguliraju razlicite ucinke u gotovo svim tkivima: kontraktilnost glatkih misica, vaskularni tonus, imunosni odgovor, nocicepciju, neurotransmisiju, embrionalni razvitak, hemostazu. Osim membranski vezanih oblika enzima, u tjelesnim tekucinama poput seruma mogu se pronaci i topljivi, enzimski aktivni oblici ekto-ATPaze. Poremecena regulacija ekto-ATPaze sudjeluje u patogenezi raznih oboljenja kao sto su degenerativni neuroloski odgovor, akutna upala, rast tumora i metastaziranje. Stoga je selektivna inhibicija te regulacija ekto-ATPaze jedno novo znanstveno podrucje koje se trenutno istražuje s velikim zanimanjem. Cilj ovog rada bio je optimirati uvjete određivanja kataliticke aktivnosti ekto-ATPaze u ljudskom serumu. Određivanje kataliticke aktivnosti ekto-ATPaze u ljudskom serumu provedeno je uz upotrebu ATP-a kao supstrata. U indikatorskoj reakciji mjerena je kolicina oslobođenog anorganskog fosfata spektrofotometrijskom metodom, s amonijevim heptamolibdatom i askorbinskom kiselinom kao reducensom, na 620 nm. Za optimiranje uvjeta koristen je ''pool'' serum te je određena kataliticka aktivnost ekto-ATPaze u 20 uzoraka zdravih osoba. Statisticka analiza provedena je upotrebom SigmaStat programa. Optimiranjem uvjeta postignuta je zadovoljavajuca aktivnost enzima na 37 °C uz 50 mmol/L HEPES-Tris pufer, pH 7, 2 koji je sadržavao 5 mmol/L CaCl2 i 3 mmol/L ATP, dok je zaustavljanje reakcije izvrseno uz 3 mol/L trikloroctenu kiselinu. Nepreciznost unutar serije određena je koristenjem "pool" seruma (N=10 ; 89, 2 ± 19, 2 nmol/mL/hr ; KV = 21, 5 %). Izmjerena kataliticka aktivnost ekto-ATPaze u 20 uzoraka zdravih osoba iznosila je 63, 7 (29, 6 - 100, 1) nmol/mL/hr. U ovom radu optimirani su uvjeti određivanja kataliticke aktivnosti ekto-ATPaze u ljudskom serumu, sto je osnova za daljnje istraživanje ekto-ATPaze u patogenezi raznih bolesti. Ecto-ATPases are transmembrane enzymes involved in hydrolysis of extracellular ATP and ADP. Extracellular ATP and other nucleotides and nucleosides are important signaling molecules that regulate a variety of effects in almost all tissues: smooth muscles contractility, vascular tone, immune response, nociception, neurotransmission, embryonic development, hemostasis. In addition to the membrane bound form of the enzyme, soluble active forms of ecto-ATPase may be found in body fluids such as serum. Dysregulation of ecto-ATPase participates in pathogenesis of various diseases (degenerative neurological response, acute inflammation, tumor growth and metastasis). Therefore, selective inhibition and regulation of ecto-ATPase is a new scientific field currently being explored with great interest. The aim of this study was to optimize conditions for determination of the catalytic activity of ecto-ATPase in human serum. The catalytic activity of ecto-ATPase in human serum was measured using the ATP as a substrate. The amount of released inorganic phosphate was measured using the spectrophotometric method, with ammonium heptamolybdate and ascorbic acid as a reducing agent, at 620 nm. "Pool" serum was used for optimization of reaction conditions, and catalytic activity of ecto-ATPase was determined in 20 healthy individuals. Statistical analysis was performed using the SigmaStat program. The satisfactory enzyme activity at 37°C was obtained with 50 mmol/L HEPES-Tris buffer, pH 7.2, containing 5 mmol/L CaCl2 and 3 mmol/L ATP, whereas the reaction was stopped with 3 mol/L trichloroacetic acid. Within-run imprecision was determined using the "pool" serum (N=10 ; 89.2±19.2 nmol/ml/hr ; CV=21.5%). The catalytic activity of ecto-ATPase measured in 20 healthy subjects was 63.7 (29.6-100.1) nmol/ml/hr. In this work conditions for determination of the catalytic activity of ecto-ATPase in human serum were optimized, which is the basis for further research of ecto-ATPase in pathogenesis of various diseases.