Biochemical variability between two Egyptian <i>Stenodactylus </i>species (Reptilia: Gekkonidae) inhabiting North Sinai

doi:10.4236/abb.2013.411129

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Advances in Bioscience and Biotechnology, 2013, 4, 974-978 ABB

http://dx.doi.org/10.4236/abb.2013.411129 Published Online November 2013 (http://www.scirp.org/journal/abb/)

Biochemical variability between two Egyptian

Stenodactylus species (Reptilia: Gekkonidae) inhabiting

North Sinai

Mohamed A. M. Kadry1*, Sayed A. M. Amer1,2

1Department of Zoology, Faculty of Science, Cairo University, Cairo, Egypt

2Department of Biology, Faculty of Science, Taif University, Taif, Saudi Arabia

Email: *[email protected]

Received 24 August 2013; revised 24 September 2013; accepted 15 October 2013

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

properly cited.

ABSTRACT

Polyacrylamide gel electrophoreses for malate dehy-

drogenase (Mdh) and beta-esterase (β-Est) isoenzymes

were conducted for biochemical differentiation be-

tween two Stenodactylus gekkonid species inhabiting

North Sinai of Egypt. Total lipids and proteins of

liver and muscle tissues in both species were also

analyzed. A total of three Mdh isoforms were re-

corded in the analysis, in which the activity of Mdh-2

and Mdh-3 seemed to be higher in S. petrii than in S.

sthenodactylus. This high activity could be supported

by the significant increase in the total lipids and pro-

teins in liver and muscle tissues of the species. It may

thus be reasonable to suppose that S. petrii is more

active, energetic and adaptable in the desert habitat

than S. sthenodactylus. β-Est showed six fractions in S.

petrii and only one fraction in S. sthenodactylus. It is

therefore noticeable that β-Est is more highly ex-

pressed in S. petrii than in S. sthenodactylus.

Keywords: Electrophoreses; Physiological Ecology;

Geckos; Isoenzymes; Lipids; Proteins

1. INTRODUCTION

Gekkonidae are among the lizards known for their strik-

ing range of morphological characteristics, ecological

habitats and body sizes. In Egypt, most of the gekkonid

species are found in and around human habitation. Some,

however, live in Egyptian deserts [1].

Many studies determined the relationships among

members of the family Gekkonidae on the basis of mor-

phological and environmental characteristics [2,3], kar-

yotyping [4,5], and biochemical [6-8] and molecular

variations [9-19]. The genus Stenodactylus contains 13

recognized species. The species Stenodactylus stheno-

dactylus and S. petrii are distributed in Egypt, Iran, Iraq,

Syria, Jordan and Arabian Peninsula [1], and areas from

Sudan to Mauritania [20].

Isoenzymes are multiple forms of a single enzyme.

The forms are often marked by different isoelectric

points and hence separable by electrophoresis. Malate

dehydrogenase (Mdh) is considered as one of the most

extensively studied isozyme systems [21]. This enzyme

with lactate dehydrogenase constitutes a very suitable

system for studying several metabolic, genetic, ecologi-

cal features, and they are very useful in systematic stud-

ies. As a homodimeric enzyme, Mdh is well known for

the many cell compartment-specific isoenzymes that cha-

racterize various organisms. There is a mitochondrial

Mdh functioning in the tricarboxylic acid cycle which is

usually NAD+-dependent. Most eukaryotes that have

been studied also have a cytosolic Mdh isoform. The

cytosolic Mdh, also known as NADP-malic enzyme

(ME), catalyzes the NADP dependent oxidative decar-

boxylation of malate into pyruvate and carbon dioxide to

generate NADPH. ME is thought to be a key enzyme in

lipid biosynthesis [22,23]. Esterase isoenzymes (Est)—as

one kind of the lipid-hydrolyzing enzymes—possess

high significance in genetics and toxicology [24]. The

present study aims to investigate the patterns of the inter-

specific biochemical variations between two common

gekkonid species (S. petrii and S. sthenodactylus) inhab-

iting the Sinai desert of Egypt.

2. MARERIALS AND METHODS

2.1. Taxon Sampling and Study Area

Collected for this project were a total of 12 individuals

*Corresponding author.

OPEN ACCESS

M. A. M. Kadry, S. A. M. Amer / Advances in Bioscience and Biotechnology 4 (2013) 974-978 975

from, firstly, 2 Egyptian gekkonid species of S. stheno-

dactylus and S. petrii and, secondly, Beer El-Abd (North

Sinai) [31˚01'2.46''N 33˚00'40.35''E] (Figure 1).

2.2. Sample Preparation and Isoezyme Assay

Tissue samples of liver and heart were taken to the lab

immediately after their removal and stored at −80˚C for

further laboratory use. For isoenzyme extraction, ap-

proximately 0.5 g of tissue was homogenized in 1 mL

saline solution NaCl (0.9%) using a manual Homoge-

nizer. 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 elec-

trophorased in 10% native polyacrylamide gel as de-

scribed by Stegemann et al. [25]. After electrophoresis,

the gels were stained according to their enzyme system,

which was followed by the incubation of the appropriate

substrate and chemical solutions at room temperature in

dark for complete staining. In most cases an incubation

of about 1 to 2 hours would be enough.

For Mdh, after the completion of 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. 0.05 M Tris-HCl pH 8.5

was prepared by dissolving 0.605 g Tris in 50 mL dis-

tilled water. The pH was adjusted to 8.5 by HCl. Then

the solution was completed to 100 ml by using distilled

water [26].

(a) (b)

Figure 1. Photos of S. sthenodactylus (a) and S. petrii (b) in-

habiting Beer Al-Abd in North Sinai.

Regarding 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 would lower the pH of the gel from

8.8 to about 7, at which the reaction would proceed read-

ily. The low temperature would minimize the diffusion

of the protein within the gel). After being rinsed rapidly

in two changes of double distilled water, the gel then

stained for esterase activity and incubated at 37˚C in a

substrate solution of 100 mg β-naphthyl acetate (β-Est)

and 100 mg fast blue RR salt in 200 ml of 0.1 M phos-

phate buffer pH 6.5 [27].

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 solu-

tion, 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.

2.3. Metabolic Reserve Study

Immediately after collection, geckos were weighted to

the nearest 0.01 - 0.1 g and dissected. Pieces of liver and

thigh muscles were removed and immediately weighted

to the nearest 0.01 g. They were stored frozen at −20˚C

till use. Livers and thigh muscles were processed for the

estimation of total lipids according to the method of

Zöllner and Kirsch [28] and total proteins according to

the method of Gornall et al. [29] using a kit of Biodiag-

nostics Company.

2.4. Statistics

All gels were scanned using Gel Doc-2001 Bio-Rad sys-

tem. For isoenzymes, the bands of enzyme activity were

designated using the known system of nomenclature [30].

Each locus was assigned with an abbreviation corre-

sponding to the name of the enzyme. When multiple loci

were involved, the fastest anodal protein band was des-

ignated as Locus One, the next as Locus Two and so on.

Student t-test in the PASW package v. 20 was used to

calculate the significance difference of total lipids and

total proteins within and between species.

3. RESULTS AND DISCUSSION

Three Mdh isoforms were recorded in the two species of

Stenodactylus. The activity of Mdh-2 and Mdh-3 iso-

forms seemed to be higher in S. petrii than in S. stheno-

dactylus. Such higher activity was reflected in the thicker

and denser bands of Mdh-2 and Mdh-3 in S. petrii (Fig-

ure 2). The cytosolic Mdhs catalyzed the NADP de-

pendent oxidative decarboxylation of malate into pyru-

vate and carbon dioxide to generate NADPH [22,23].

Due to its ability to produce NADPH, this enzyme is

thought to be a key enzyme in lipid biosynthesis [23].

The apparent increase in the activity of Mdh in liver

M. A. M. Kadry, S. A. M. Amer / Advances in Bioscience and Biotechnology 4 (2013) 974-978

976

storage or enzyme activity. tissues of S. petrii, in the present study, could be sup-

ported by the significant increase in the total lipids and

proteins in liver and muscle tissues of this species. This

species is also shown to be fattier than S. sthenodactylus.

It is thus possibly reasonable to consider S. petrii more

active, energetic and adaptable in the desert habitat than

S. sthenodactylus.

The present results revealed higher activity of es-

terases in the examined tissues of S. petrii than in S.

sthenodactylus. Esterases are used as bio-indicators to

measure the toxic potency of pesticide residues [31]. The

presence of only one isoform of esterases, β-Est-1, in

heart tissue of S. sthenodactylus may—to some extent—

reflect the safety of the diet applied to this animal in the

field , which is unlike the case with that for S. petrii [31].

β-Est showed six fractions in S. petrii and only one

fraction in S. sthenodactylus. These fractions were denser

and thicker in S. petrii (Figure 3). The first fraction in

the S. sthenodactylus was the only clear fraction in the

electrophoretic pattern, while the second fraction was

recorded only in two samples of this species. It is there-

fore noticeable that β-Est is highly expressed more in S.

petrii than in S. sthenodactylus. No reasonable explana-

tion has been found as regards why many bands disap-

peared in the pattern of S. sthenodactylus. But factors can

be supposed, such as the staining reaction, sampling

Table 1 records the mean and standard error values of

the total lipids and proteins in the liver and muscle tis-

sues of both Stendoda ctylus species. By comparing the

total lipids and total proteins of liver and muscle tissues

in the two Stenodactylus species, we found more signifi-

cant increase in the total lipids in the liver (P < 0.001)

and muscle (P < 0.01) tissues of S. petrii than in those of

S. sthenodactylus. S. petrii also showed more significant

increases (P < 0.01) in total proteins in liver and muscle

1 2 3 4

789



1011 12

dh-1

dh-2

dh-3

Figure 2. The electrophoretic profile of Mdh isoenzymes in liver tissues. Lanes are as follows: 1-6

(S. petrii), 7-12 (S. sthenodactylus).

12 3 456789



10 11 12

-Est-1

-Est-2

-Est-3

-Est-4

-Est-5

β-Est-6

Figure 3. The electrophoretic profile of β-Est isoenzymes in heart tissues. Lanes are as follows:

1-6 (S. petrii), 7-12 (S. sthenodactylus).

Table 1. Comparison of total lipids and total proteins in liver and muscle tissues of S. petrii and S. sthenodactylus. Data are

expressed as mean ± standard error. Number of individuals between parentheses.

Parameters S. sthenodactylus S. petrii t-test

Liver total lipids (mg/100mg) 8.518 ± 1.209 (6) 10.384 ± 2.265 (6) 7.526***

Thigh muscle total lipids (mg/100mg) 4.250 ± 0.738 (6) 8.033 ± 3.820 (6) 3.165**

t-test 6.845*** 4.290**

Liver total protei ns (mg/100mg) 96.095 ± 31.717 (6) 216.909 ± 31.717 (6) 4.617**

Thigh muscle total proteins (mg/100mg) 57.154 ± 32.605 (6) 134.735 ± 27.767 (6) 4.078**

t-test 3.411** 5.804***

Body weight (g) 2.083 ± 0.098 (6) 4.750 ± 0.414 (6) 7.589***

Highly significant at P < 0.01. ***Very highly significant at P < 0.001.

OPEN ACCESS

M. A. M. Kadry, S. A. M. Amer / Advances in Bioscience and Biotechnology 4 (2013) 974-978 977

tissues than S. sthenodactylus. Within each species, total

lipids and proteins were significantly higher in liver (P <

0.01, P < 0.001) tissues than in muscle tissues.

4. CONCLUSION

In conclusion, S. petrii displayed higher physiological

performance and activity than S. sthenodactylus, while

isoenzyme expression was higher in the first species than

in the second. The accumulation of total lipids and pro-

teins was also significantly higher in the first species

than in the second. Data analysis in the present research

project does not support what is concluded in the study

of Amer [6] for both species and within S. petrii. We

therefore can affirm that the identification of S. petrii is

incorrect in the study by Amer [6], while the research

results point to the identity of other haplotypes of S.

sthenodactylus than that of S. petrii.

5. ACKNOWLEDGMENTS

We are grateful to Dr. Shawkat Ahmed at Ain Shams University of

Egypt for his technical support in conducting the practical part of

isoenzyme assay in this work.

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