ABSTRACT

Malate dehydrogenase, α-esterase and β-esterase isoenzymes have been analyzed by native polyacrylamide gel electrophoresis in different tissues (liver, kidney, muscle and hump) of the Arabian Camel, Camelus dromedaries in order to study the tissue specificity of these isoenzymes. Malate dehydrogenase recorded three fractions while both esterases recorded two fractions in different studied tissues. Malate dehydrogenase was considered as a key isoenzyme in discriminating among the different studied tissues. The percentage amount of both Mdh-3 and Mdh-2 showed significant difference among the different tissues while Mdh-1 recorded significant variation between muscle and hump tissues. The significant variation in the amount of Mdh isoenzyme (on the level of either total or fractions) among the different studied tissues may reflect the role of this enzyme for energy production in this desert animal. With respect to esterases, the percentage amount for both fractions and the total enzyme did not show significant difference among different tissues except β-Est-2 which was significantly higher in hump than in muscle. This enzyme showed higher thickness and intensity in both liver and kidney than in the muscle and hump indicating its greater activity in both liver and kidney.

1. INTRODUCTION

The dromedary (Camelus dromedarius), also called Arabian camel or one humped camel, was domesticated some 5000 years ago in the Arabian Peninsula. This species is able to survive in hot temperatures that is normally lethal to other species. All the functions of this species are seen to be adapted to desert environment which is characterized by little water and poor food [1]. The total population of dromedary is estimated to be around 1.6 million camels within the Arabian Peninsula constituting 51% of the total tropical livestock unit in Saudi Arabia. It is economically very important for milk and meat production which was mainly linked to the population growth [2].

The Arabian camel has paid the attention of many investigators on physiological [3-5], behavioral [6-8], genetical [9-11] and ecological [12,13] levels.

Isoenzymes are multiple forms of a single enzyme, which often have different isoelectric points and therefore can be separated by electrophoresis. Electrophoretic studies were done extensively on the different tissues of various animals from which it reveals that the enzyme exit in multi molecular forms and functions [14]. Several isoenzymes of malate dehydrogenase (EC 1.1.1.37) exist. There are two main isoforms in eukaryotic cells [15]. One is found in the mitochondrial matrix, participating as a key enzyme in the citric acid cycle that catalyzes the oxidation of malate. The other is found in the cytoplasm, assisting the malate-aspartate shuttle with exchanging reducing equivalents [16]. Esterase isoenzymes are one of the lipid-hydrolyzing enzymes, possess high significance in genetics and toxicology [14,17]. Esterases are also used as bioindicators to measure the toxic potency of pesticide residues usually applied in agriculture [18].

To the best of our knowledge, there is no study tackling the isoenzyme system and its tissue specificity in the Arabian camel so far. The present study therefore, aimed to investigate the patterns of variations of malate dehydrogenase and esterases (α and β) isoenzymes in different tissues of the Arabian camel that has been used for milk and meat production.

2. MATERIALS AND METHODS

2.1. Sample Preparation and Isoenzyme Assay

Tissue samples of liver, kidney, muscle and hump were freshly obtained from a slaughterhouse in Taif city, immediately taken to the lab and stored at −80˚C for further laboratory use.

For isoenzyme extraction, approximately 0.5 g of tissue was homogenized in 1 mL saline solution NaCl (0.9%) using a manual Homogenizer. The homogenates were centrifuged at 5000 rpm for 10 minutes and the supernatants were kept at −20˚C until use. For electrophoresis, 30 μL of the extract was mixed with 10 μL of treatment buffer and 35 μL of this mixture was applied to the well. Isoenzymes were electrophorased in 10% native-polyacrylamide gel as described by Stegemann et al. [19]. After electrophoresis, the gels were stained according to their enzyme system with the appropriate substrate and chemical solutions then incubated at room temperature in dark for complete staining. In most cases the incubation for about 1 to 2 hours is enough.

2.2. Malate Dehydrogenase (Mdh)

After electrophoresis, the gel was soaked in 100 mL of 0.05 M Tris HCl pH 8.5 containing 25 mg NBT, 25 mg EDTA, 25 mg NAD, 10 mg malic acid and 3 mg PMS [20]. 0.05 M Tris HCl pH 8.5 was prepared by dissolving 0.605 g Tris in 50 mL distilled water. The pH was adjusted to 8.5 by HCl. Then the solution was completed to 100 ml by distilled water.

2.3. α and β Esterase Enzymes (α-Est and β-Est)

After electrophoresis, the gel was soaked in 0.5 M borate buffer (pH 4.1) for 90 minutes at 4˚C. This procedure lowers the pH of the gel from 8.8 to about 7 at which the reaction proceeds readily. The low temperature minimizes diffusion of the protein within the gel. The gel then was rinsed rapidly in two changes of double distilled water. The gel was stained for esterase activity by incubation at 37˚C in a substrate solution of 100 mg α- naphthyl acetate (α-Est) and b-naphthyl acetate (b-Est) and 100 mg fast blue RR salt in 200 ml of 0.1 M phosphate buffer pH 6.5 [21].

2.4. Gel Fixation

After the appearance of the enzyme bands, the reaction was stopped by washing the gel two or three times with tap water. This was followed by adding the fixative solution, which consists of ethanol and 20% glacial acetic acid (9:11 v/v). The gel was kept in the fixative solution for 24 hours and then was photographed.

2.5. Statistics

All gels were scanned using Gel Doc-2001 Bio-Rad system. For isoenzymes, the bands of enzyme activity were designated using the known system of nomenclature [22]. An abbreviation which corresponds to the name of the enzyme designated each locus. When multiple loci were involved, the fastest anodal protein band was designated as locus one, the next as locus two and so on. Student t-test in the SPSS package v. 13 was used to calculate the significant differences of the percentage amounts for isoforms among the different studied tissues.

3. RESULTS AND DISCUSSION

3.1. Malate Dehydrogenase

Table 1 showed the mean and standard error for the per-