Advances in Bioscience and Biotechnology, 2013, 4, 974-978 ABB Published Online November 2013 (
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: *
Received 24 August 2013; revised 24 September 2013; accepted 15 October 2013
Copyright © 2013 Mohamed A. M. Kadry, Sayed A. M. Amer. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
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
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.
2.1. Taxon Sampling and Study Area
Collected for this project were a total of 12 individuals
*Corresponding author.
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.
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
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M. A. M. Kadry, S. A. M. Amer / Advances in Bioscience and Biotechnology 4 (2013) 974-978
Copyright © 2013 SciRes.
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
1011 12
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
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.
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.
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.
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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