Estimation of 5`-nucleotidase activity from saliva of diabetic patients with partial purification of its isoenzymes

Abstract: This study was performed on (60) saliva specimens, including (30) diabetic patients in addition (30) healthy saliva specimens were taken from healthy subjects as control group. The results showed a significant increase in the activity of saliva 5`-nucleotidase (5`-NT) from diabetic patients compared to healthy subjects. This study included a partial purification of 5`-NT as well, using (35%) (NH 4 ) 2 SO 4 for precipitation and gel filteration by (sephadex G-75 ) which gave single peak. Then 5-NT was partially also purified by using Ion exchange chromatography ( DEAE-cellulos A-50), in which three isoenzymes were isolated with different degree of purity. Introduction: 5`-nucleotidase (5`-NT) is a membrane glycoprotein enzyme that is found as a widest scope in mammalian cells (1) , animals, plants , parasites and in human body (2) . 5`-nucleotidase or nucleoside mono phosphate phosphohydrolases (EC 3.1.3.5) is an enzyme catalyzing the hydrolytic dephosphorylation of nucleoside monophosphate to nucleoside and orthophosphate [AMP + H 2 O→Nucleoside + PO 4 -3 ] (3,4) . The prescence of 5`-NT in normal human erythrocytes is highly specific with pyrimidine and not purine nucleotide as substrate. The physiological role of nucleotidase is not well understood but may include the provision of nucleoside for cellular uptake and the enzyme may play a modulating role in ATP and ADP regulatory action at the cell surface (5) , and it participates (as catabolic enzyme) in the regulation of nucleotide level in living cells (6,7) . 5`-NT is associated with rebuilding DNA (DNA turn-over) , multiply in bioprocess pathway of pyrimidine and purine (8) . There are two types of 5`-NT isoenzymes; cytoplasmic isoenzyme (soluble in cytoplasma) and associated with wall called (Ecto-5-Nucleotidase) related to outer surface of the cell. The exoenzyme associated with metabolic process of nucleotides (4) , while cytoplasmic isoenzyme associated with adenosine tri phosphate (ATP) hydrolysis in cell to adenosine or IMP to inosine ,therefore cytoplasmic enzyme regulates hydrolysis of inner nucleotides (9) . 5`-NT isoenzymes were purified from several sources such as rat liver, in which two isoenzymes have been isolated (10) ,and many isoenzymes have been isolated from kidney rat and mouse by electrophoresis (11) . Two 5`-NT isoenzymes separated and purified from human serum (12) and from liver sheep (13) , while Al-Salihi et al , (2009) purified three isoenzymes from serum of anemic patients (14) . 5`-NT has a great clinical importance clinically and remedially, Its significance is considered for a lot of morbidity cases. So the a available information about 5`-NT is useful to diagnose or to treat the disease. The use of saliva as a diagnostic tool for a lot of diseases has been expanded , this study aimed to estimate the activity of 5`-NT in saliva of normal and pathological conditions such as diabetic patients in order to evaluate its diagnostic significance, and to partial purification of 5`-NT isoenzymes from saliva of diabetic patients. Materials & Methods A study was conducted taking saliva of diabetic patients, cases subjects were divided into two groups ; case group I , it included (30) samples from diabetic patients at (14-80) years age . Cases group II (control) , it included (30) samples from healthy subject at (18-70)years age. Enzyme assay: 5`-NT activity was measured according to Fisk and Sobbarow method (15) , using inorganic phosphate as standard. 5`-NT activity is defined as µmol inorganic phosphate that produced from substrste 5`-AMP by Fisk and Sobbarow reagent. Estimation of protein: Protein contents were estimated by the method of lowry (16) with bovine serum albumin (BSA) as standard. Enzyme purification: The purification of 5`-NT included several steps described as below: - Ammonium sulphate option : (35)% Ammonium sulphate was added to the saliva (salting out) and the solution centrifuged at (3500rpm). The pellet was suspended in (3.5)ml of (50)mM Tris-HCl buffer (pH 7.2) and dialysed against the same buffer for overnight with continues changing of the buffer. - Gel filtration on sephadex G-75 : (1.5)ml from isolated 5`-NT above was loaded on sephadex G-75 (30 × 2cm) , which was swollen in (50)mM Tris-HCl buffer (pH 7.2 ), and the 5`-NT was eluted with (50) mM Tris-Hcl buffer pH 7.2, and collected into different fractions (5)ml. - Ion Exchange Chromatography : (2.5)ml eluant from gel filtration column above was loaded on DEAE Cellulose A-50 column (ion exchange chromatography ) (30 × 1.5)cm , which was swollen in (50)mM Tris-HCl pH 7.2 with using NaCl gradient (0.02-0.4)M , Eech fraction collected (10) ml. Statistical analysis : Values were calculated as mean±SD and the statistical analysis was done using SPSS 17.0 software. Student unpaired t-test was used for comparison between two groups. The p-value of