Phosphorylase from muscle of tilapia (Tilapia mosambica) was extracted by water and purified by ammonium sulphate precipitation, centrifugation and repeated recrystallisation. Electro-phorogram of the enzyme preparation showed a single band near origin. The enzyme showed optimum pH and temperature at 6.1 and 37°C respectively. Glucose and glucose-6-phosphate were found to be competitive inhibitors of the enzyme. Maltose and starch acted as primers for the  phosphorylase reaction like glycogen

Mukundan, M.K.

Nair, M.R.

Aquatic Commons

p E 
E F SCLE 
PI (TILAP!A MOSAMBICA) 
~K.MUKUNDAN AND M.R.NAIR 
Central Institute of Fisheries TechrJologv, Willingdon Island, Cochin-682003. 
Phosphorylase from muscle of tilapia (Tilapia mosambica) 
was extracted by water and purified by ammonium sulphate 
precipitation, centrifugation and repeated recrystallisation. Electrc-
phorogram of the enzyme preparation shovved a single band 
near o~igin. The enzyme showed optimum pH and temperature 
at 6.1 and 37°C respectively. Glucose and glucose-6-phosphate 
were found to be competitive inhibitors of the enzyme. Maltose 
and starch acted as primers for the phosphorylase reaction 
like glycogen. 
INTRODUCTION 
Fish muscle resembles mammalian 
muscle in many respects. Muscle glyco-
gen is an important source of energy for 
fish during muscular activity when glycogen 
is degraded to lactic acid via the EMP 
pathway (Burt, 1961 and 1966; Burt and 
Stroud, 1966; Tarr and Leroux, 1962 and 
Nagayama, 1961). Fish muscle contains 
the enzymes required to bring about this 
oxidative degradation as in mammalian 
muscle. Fish muscle is a source of both 
glycogen and fat of which glycogen is 
used mainly for muscular activity as is 
evident from the depletion of glycogen 
in struggled fish (Edith Gould, 1965) while 
fat is used mainly during spawning season 
and starvation. In insects, glycogen is 
VoL 14 No. 1 1977 
used only for flight initiation and then 
changes over to fat for prolonged mus-
cular activity (Opie and Newsholme, 1967 
and Applebaum and Schlesinger, 1973). 
This is in contrast to the predominant 
carbohydrate utilisation in mammals where 
fat is a fuel reserve to be used during 
starvation. 
The enzyme phosphorylase involved 
in reversible conversion of glucose-1-
phosphate to glycogen has been studied 
by Burt (1966) in crude extracts of cod 
and by Crabtree and Newsholme (1972) 
in certain fishes, crustaceans and frog. 
So far, there is no account of purification 
and characterisation of muscle phospho-
rylase from fish. Many compounds are 
known to affect phosphorylase with vari-
1Y1uliunda.n & Nair: Pfwspfiorylase from ti!apia mw;cle 
ations depet1ding upon the source of 
enzyme. The present paper reports the 
results of experiments carried out to purify 
and characterise muscle phosphorylase of 
tilapia. 
MATERIALS AND METHODS 
Glycogen, Glucose-1-phosphate dipot-
assium salt, glyceraldehyde-3-phosphate 
dipotassium salt and glucose-6-phosphate 
dipotassium salt (L. Light and Company, 
England) and adenosine monophosphate 
(AMP) (Fluka, Switzerland) were repeatedly 
recrystallised from ethanol water mixture 
before use, except glycogen, to free from 
traces of inorganic phosphate and the 
purity was confirmed by paper chroma-
tography. The reagents for polyacrylamide 
gel electrophoresis were the products of 
BDH, England except tris hydroxymethyl 
amino methane which was a product of 
Fluka. All other chemicals were of analar 
quality. A refrigerated high speed centri-
fuge (International Equipment Company, 
Model HR-1) was used for centrifugation. 
Evelyn colorimeter (Honeywell Rubricorn 
Instruments) was used for colorimetric 
measurements. 
Tilapia weighing 300 to 500 g. caught 
from fish farms at Malampuzha and Chi-
ttoor and maintained live in aquarium 
tanks were used: Both male and female 
fish were used indiscriminately. 
Purification of the enzyme 
Fish were caught and killed imme-
diately by a blow on the head to 
avoid struggling as far as possible. They 
were skinned and the lateral muscle 
excised and 500 g. muscle used for the 
study. AU operations, unless otherwise 
stated, were carried out in a cold room 
maintained at 0 to 4°C. "Phosphorylase 
was then extracted according to the method 
of Cori (l955), unless otherwise specified. 
The buffer used for dialysis had the com-
position of 25 ml. 0.015M. ethylenediamine 
tetra acetic acid (EDTA) and 1000 ml. 
0.02 M. Sodium /3 glycerophosphate and 
the mixture diluted with distilled water 
to 2500 ml. and pH adjusted to 6.8. The 
solvent for the enzyme crystals was com-
posed of 2 ml. 0.0375 M. EDTA and 
48 mi. of 0.02 M. sodium j3 glyceropho-
spl:ate having a pH 6.8. All pH manipu-
latiOns were effected with 0 05 N hydro-
chloric acid to the acidic side and saturated 
potassium bicarbonate solution to the 
alkaline side. The dialyses were conducted 
in cellophane bags 1.3 em. diameter at 
0 to 4°C. 
Enzyme assay 
The enzyme activity was assayed in 
the direction of glycogen synthesis as 
described by Crabtree . and Newsholme 
(1972). The assay medium contained 32m.M. 
glucose-1-phosphate, 0.5 m. M. AMP, 
TABLE I 
SUMMARY OF PURIFICATION 
Fraction Total activity Sp. activity Percentage of recovery 
Crude 13,20:) 0.0878 
First crystals 8,700 25.87 69.9 
Second crystals 5,525 35.65 41.5 
Third crystals 4,520 38.39 34.3 
2 Fish. Techno!. 
MufLmdan & Nair: Pftospftorylase from tilaj)ia muscle 
2% (w/v) glycogen and pH adjusted to 
6.2. The reaction was initiated by the 
addition of 0.2 mi. of the enzyme pre-
paration to 0.2 mi. of the assay medium 
in a polystyrene tube kept incubated at 
36°C for 15 minutes. The reaction was 
terminated by the addition of 0.6 mi. 
perchloric acid. The protein precipitated 
was removed by centrifugation and in the 
centrifugate the inorganic phosphate libe-
rated was estimated by the method of 
Fiske and Subbarow (1925). The a~say 
is based on the assumption that during 
this reaction the liberation of inorganic 
phosphate is linear. AMP and glucose-1-
phosphate were omitted from the control. 
Protein concentrations were determined 
by the method of Follin and Ciocaltue 
(1927). 
RESULTS 
Purification 
Table I shows the summary of puri-
fication of tilapia muscle phosphorylase. 
An enzyme unit is defined as the amount 
of enzyme in mg. that liberates one micro-
mole of inorganic phosphate per minute 
under optimum conditions of temperature 
and pH. Specific activity is defined as 
the number of units of enzyme per mg. 
of protein. 
Effect of pH 
The effeci: of pH was studied by 
carrying out the phosphorylase reaction 
for 15 minutes in l mi. citrate buffer as 
given in assay methods and then merging 
the results with those from trismaleate 
buffer. Muscle phosphorylase of tilapia 
recorded only one pH optimum at 6.1 
(Fig. 1) as against two pH optima at 5.5 
and 6.7 for cod muscle phosphorylase 
VoL 14 No. 1 1977 
03 \ 
\ 
().{ 
Fig. 1 
Effect of pH on Tilapia muscle phospho-
rylase activity. The assay conditions 
were as described under materials 
and methods. 
(Burt, 1966), 6.2 for mammalian muscle 
phosphorylase (Sumner and Myrback, 1951) 
and 6.7 for fat body phosphorylase of 
locusts (Applebaum and Schlesinger, 1973). 
Effect of temperature 
The reactions were carried out at 
temperatures varying from 20 to 55°C for 
15 minutes. The activity showed the 
maximum value at 36°C, almost similar 
to mammalian enzyme. The enzyme lost 
most of its activity above 50°C. These 
results are indicated in Fig. 2. 
Substrate specificity 
For study of substrate specificity, 
0 2 ml. each of saturated solutions of 
gl ucose-1-phosphate and glucose-6-phos-
phate were separately incubated at 36°C 
with 0.2 mi. of a solution containing 2/~ 
3 
Nh&undar.1 & Na~r: Pftospfwrylase from tilapia muscle 
2·0 
= 1·5 -
E 
"' :::
0. 
~ 
0 
c 
E 
:<:'O 
i:: g 
-' w 
> 0·5 
0 10 
Fig.:!:. 
I~ 
1 
/ 
~ 
20 30 
TEMPERATURE 
Fig. 2 
40 50 60 
Effect of temperature of Tilapia muscle 
phosphorylase. The assay conditions 
were as described under materials 
and methods. 
glycogen and 0.5 m. M. AMP for 30 
minutes. At the end of 30 minutes, the 
reaction mixture was heated to 50°C to 
inactivate the enzyme. Then the reaction 
mixtures were chromatographed on paper 
along with standards for glucose-l-phos-
phate, glucose-6-phosphate, AMP and 
dipotassium hydrogen phosphate. They 
were run with butanol : acetic acid : etha-
nol : water in the proportion of 120 : 
50 : 25 : 25 for 20 hours and developed 
with phosphate reagent (Ivor Smith, 196eJ). 
Inorganic phosphate was released only 
from the reaction mixture that contained 
glucose-1-phosphate showing that tilapia 
muscle phosphorylase is sp~cific for L-D. 
glucose-1-phosphate. Studies were also 
carried out in a similar way to see the 
nature of primers to which tilapia muscle 
phosphorylase can add on glucose units. 
4 
The results indicated that apart from 
glycogen, maltose and starch had the capa~ 
city to act as primers, but not dextrin. 
Inhibition 
Glucose and glucose-6-phosphate are 
competitive inhibitors of tilapia muscle 
phosphorylase for the substrate glucose-
1-phosphate. The rate of inhibition varied 
with increase in concentration of substrate 
in both cases. AMP relieved the inhibition 
by glucose-6-phosphate as recorded by 
Kamagawa and Fukui (1971) in disagre-
ement with the report of Applehaum and 
Schlesinger (1973) (Fig. 3). 
Fig. 3 
The effect of inhibitors, glucose and 
glucose-6-phosphate on tilapia 
muscle phosphorylase 
0 without inhibitor 
D with 25 m.M. Glucose inhibitor 
x with 15 m.M. Glucose-6-phosphate 
inhibitor 
Assay conditions as given under 
materials and methods 
Effect of primer concentration 
The velocity of tilapia muscle phos-
phorylase for a given concentration of 
enzyme increased with the increase m 
Fish. Techno!. 
Mufmndan & Naii·~ Pftospfiorylase from tilopia muscle 
,, 
.c;•N."-N .,...t/.J..'f(:CU. • .k.H 
~~ (FA.!OO""'/ 
Fig. 4 
Influence of glycogen concentration on 
phosphorylase reaction. Assay conditions 
as given under materials and methods. 
Specific activity of the enzyme 3. 839. 
concentration of glycogen. For an enzyme 
preparation with specific activity 38.39 
the maximum activity was recorded for 
a glycogen concentration of lg. per cent. 
Further increase in glycogen concentration 
had no effect on the velocity of phospho-
rylase reaction. (Fig. 4). 
DISCUSSION 
The function and distribution of glyco-
lytic enzymes are technologically important. 
The fact that the breakdown products 
of carbohydrate contribute to flavour, 
taste and texture of meat in general str-
esses this point. Further, the enzymes 
involved in carbohydrate breakdown play 
an important role in rigor mortis, since 
the amoun.t of lactic acid formed during 
the premortem activity influence the time 
for its onset and duration, (Tomlinson, 
Jonas and Geiger, 1963 and Burt, 1966) 
along with adenosine triphosphate con-
centration. 
VoL 14 No. l 1977 
Tilapia muscle phosphorylase is similar 
to mammalian muscle in several respects. 
The optimum temperature and pH are 
almost identical to the mammalian enzymes 
reported by Cori (1955). Though AMP 
activates fish muscle phosphorylase, it is 
not essential for fresh preparations of the 
enzyme, showing the· existence of active 
'a' and inactive 'b' forms of phosphory-
lase in tilapia muscle as in cod (Burt, 
1966 a) and mammals (Cori, 1955). In 
this respect tilapia muscle pho~phorylase 
is different from protozoan and yeast 
phosphorylase whose activity is fully in-
dependent of the presence or absence of 
AMP (Applebaum and Schlesinger, 1973). 
The extent of inhibition by glucose and 
the capacity of AMP to relieve it forms 
another similarity of tilapia muscle phos-
phorylase to mammalian muscle phospho-
rylase. Above all like phosphorylases 
from all sources tilapia muscle phospho-
rylase is specific for the substrate L-D-
glucose-1-phosphate. The main difference 
between mammals and tilapia as regards 
the enzyme content is its thrt>e fold con-
tent in the former than in the latter. 
But when compared to insects and cru-
staceans (Opie and Newsholme, 1972 and 
Applebaum and Schlesinger, 1973) tilapia 
contains a larger proportion of the enzyme. 
Thus tilapia is in between mammals, and 
insects and crustaceans, when the extent 
of glycolysis is considered. 
The inhibition by glucose and glu-
cose--6-phosphate explains the preferred 
use of them over the polysaccaride gly-
cogen, which are comparatively a quick 
source of energy in glycolysis. The inhi-
bitory effects of these compounds will be 
transcicnt in vivo due to its oxidation in 
EMP pathway, that it can only be a case 
of product inhibition. Thus intermediates 
5 
of glycolytic pathway cannot act as a 
standing inhibitor in vivo. So, for any 
attempt to suppress glycolysis an end pro-
duct of the carbohydrate metabolism will 
be of better use than the glycolytic in-
termediates. 
REFERENCES 
Applebaum, S. W. and H. M. Schlesinger. 
1973. Biochem. J., 135 : 37. 
Burt, J. R. 1961. J. Fd. Sci., 26 : 462. 
Burt, J. R. 1966. J. Fish. Res. Bd Can .• 
13, 4 : 527. 
Burt, J. R. and G. D. Stroud. 
Bull. lap. Soc. Scient. Fish., 
2: 204. 
1966. 
32, 
Cori, C. T. 1955. Methods of Enzymolo-
gy., I : 200. Academic Press, New 
York. 
Crabtree, B. and E. A. Newsholme. 1972. 
Biochem. J., 126 : 49. 
Edith Gould. 1965. Testing the freshness 
of frozen fish: 14, Fishing News. 
(Books) Ltd., London. 
6 
Fiske, C. H. and Y. Subbarow. 1925. J. 
Biol. Chern., 66 : 375. 
Folin, 0. and V. Ciocaltue. 1927. J'. 
Biol. Chern., 131 : 627. 
Ivor Smith. 1960. Chromatographic and 
Electrophoretic Techniques, I : 231, 
William Hineman Medical Books Ltd .• 
London. 
Kamagawa, A. and Fukui, T. 1971:. 
Biochim. Biophys. Acta., 242 : 55. 
Nagayama. 1961. Bull. J'ap. Soc. Scient. 
Fish., 27, 11 : 1022. 
Opie, L. H. and E. A. Newsholme. 1967. 
Biochem. J., 103 : 391. 
Sumner and Myrback. 
I, Part H : l02L 
New York. 
1951. Enzymes., 
Academic Press, 
Tarr, H. L. A. and M. Leroux. 1962. 
Can. J. Biochem. Physiol., 40: 57L 
Tomlinson, N. R., E. E. Jonas and S. E. 
Geiger. 1963. J. Fish. Res. Ed Can., 
10, 5: 1145. 
Fish. Techno!. 


Purification and characterisation of phosphorylase from muscle of Tilapia (Tilapia mosambica)

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Purification and characterisation of phosphorylase from muscle of Tilapia (Tilapia mosambica)

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