Title of Invention

A PROCESS FOR INHIBITING THE ADENOSINETRIPHOSPHATASE (ATPASE) ACTIVITY OF FISH ACTOMYOSIN USING PLOYHYDRIC ALCOHOLS

Abstract Abstract A process for inhibiting the adenosinetriphosphatase(ATPase) activity of fish actomyosin using polyhvdric alcohols The present invention relates to a process for inhibiting the adenosinetriphosphatase(ATPase) activity of fish actomyosin using polyhydric alcohols. More particularly it relates to the inhibition of nucleotide degrading enzyme adenosinetriphosphatase (ATPase) from the sardine (Sardinella longiceps) actomyosin by sorbitol and mannitol. Freezing and frozen storage is the most widely accepted method of preservation of muscle proteins. During this process denaturation /aggregation of myofibrillar proteins results in the loss of functionality of proteins. Various cryoprotectants and its combinations have tried to reduce the denaturation during freezing (Sultanbawa and Li-Chan 1998, Cryoprotective effect of sugars and polyol blends in ling cod surimi during frozen storage. Food Research International, 31(2): 87-98). Myosin is the major contributor of the functionality of muscle food and it has been extensively studied for improving the functionality by various treatments (Kristinsson and Hultin,2003. Effect of low and high phi treatments on the functional properties of cod muscle proteins. J. Agri.. Food Chem. 51: 5103-5110; Kristinsson and Hultin,2003b. Changes in the conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding. J. Agric. Food Chem. 51: 7187-7196).
Full Text Field of invention
The present invention relates to a process for inhibiting the adenosinetriphosphatase(ATPase) activity of fish actomyosin using ployhydric alcohols. More particularly it relates to the inhibition of nucleotide degrading enzyme adenosinetriphosphatase (ATPase) from the sardine {Sardinella longiceps) actomyosin by sorbitol and mannitol.
Background of invention
Freezing and frozen storage is the most widely accepted method of preservation of muscle proteins. During this process denaturation /aggregation of myofibrillar proteins results in the loss of functionality of proteins. Various cryoprotectants and its combinations have tried to reduce the denaturation during freezing (Sultanbawa and Li-Chan 1998, Cryoprotective effect of sugars and polyol blends in ling cod surimi during frozen storage. Food Research International, 31(2): 87-98). Myosin is the major contributor of the functionality of muscle food and it has been extensively studied for improving the functionality by various treatments (Kristinsson and Hultin,2003. Effect of low and high pH treatments on the functional properties of cod muscle proteins. J. Agric. Food Chem. 51: 5103-5110; Kristinsson and Hultin,2003b. Changes in the conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding. J. Agric. Food Chem. 51: 7187-7196).
Myosin is a highly asymmetric molecule which has molecular size of 520 kD consists of two heavy chains and two pairs of light chains (Rayment et a/., 1993. Three dimensional structure of myosin subfragment-1: a molecular motor. Science 261: 50-58). Myosin is one of the principal protein components of numerous contractile systems and comprises almost 50% of the total protein in skeletal muscles (Harrington and Rodgers,1984 {Myosin. Ann. Rev.Biochem, 53: 35-73). The N-terminal half of each heavy chain folded into a globular head or myosin subfragment 1 (S1) which contains the actin and ATP binding sites(Togashi et a/.,2002. Differential scanning calorimetry and circular dichroism spectrometry ofwallye Pollack myosin and light meromyosin. J.Agric. Food Chem.

50: 4803-4811). Adenosinetriphosphate is enzymatically degraded through a series of intermediates to hypoxanthine in sea food, resulting in rigor mortis, a decline in muscle pH and poor quality meat (Ashie et a/., 1996. Spoilage and shelf life extension of fresh fish and shell fish. Critical Reviews in Food Science and Nutrition 36: 87-121). The faster the rate at which muscle were frozen the slower would be the rate of postmortem glycolysis and of ATP break down and that this were to be reflected in less exudation on thawing. Magnesium sulfate was injected pre-slaughter in dosed sufficient to cause relaxation of the animal, the rate of ATP break down was slowed down even more and the retention of in vivo water holding capacity was enhanced to an even greater extent and is reflected in extent of drip. When ATP breakdown was arrested then the degree of drip on thawing would be less still (Lawrie, R.A., 1968 recent advances in food science volume 4 p.365).
Following the death of fish, the conversion of ATP to IMP is usually complete within 1 or 2 days and this is presumed to be totally catalytic process. The formation of hypoxanthine from ATP is fast and depends on endogenous enzymes. The post mortem depletion of ATP and ADP permits actin and myosin to interact, forming permanent actomyosin cross links (Ashie ef a/., 1996 Spoilage and shelf life extension of fresh fish and shell fish. Critical Reviews in Food Science and Nutrition 36: 87-121).
The nucleotides ATP, ADP and IMP have been shown to exert a protective effect on fish actomyosin stored at -20 °C. While nucleotide catabolites ionosine and hypoxanthine destabilized these proteins (Jiang et a/., 1987. Effect of adenosine nucleotides and their derivatives on the denaturation of myofibrillar proteins in vitro during frozen storage at-20°C. J Food Sci. 52(1): 117-123). This finding may help explain why fresh fish with consequent higher concentrations of ATP, ADP and IMP are most stable during frozen storage than less fresh fish (Fukuda et a/., 1984. Effect of freshness of chub mackerel on the freeze denaturation of myofibrillar proteins. BullJap.Soc.Sci.Fish 50: 845-852).
Reference may be made to a number of compounds which inhibits the sarcoplasmic reticulum ATPase activity. Ca2+ and Mg2+ ATPase activity of skeletal

muscle sarcoplasmic reticulum was inhibited by nonyiphenol and 3,5 dibutyl-4-hydroxy toluene (BHT) (Michelangeli,S. et a/., 1990. Mechanism of inhibition of the (Ca2+Mg 2+) ATPase by Nonyiphenol. Biochemistry 29: 3091-3101). Ca2+ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by 2,5, -di-tert-butyl-1,4,-dihydroxybenzene (BHQ) and other hydrophobic 1,4-dihydroxy benzenes ( Khan, et a/., 1995. Interactions of dihydroxy benzenes with the Ca2"ATPase: separate binding sites for dihydroxy benzenens and sesquiterpene lactones. Biochemistry,34: 14385-14393). A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibitioin has been found . Phospholamban decreases the number of activable Ca2+ATPase without affecting substrate affinity or the ability of nucleotides to serve its dual modulatory roles i.e. catalytic and regulatory. Tannin even under stimulatory conditions is a competitive inhibitor of Mg2+ ATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentration results from competition of tannin with the nucleotide-binding site of Ca2+ATPase. In contrast ellargic acid produced a curvilinear Dixon plot suggesting of partial inhibition of nucleotide activation (Coll.K.E., et a/.,1999. Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2* adenosinetriphosphatase. Biochemistry 38: 2444-2451).
Thapsigargin, a plant derived sesqiterpene lactone, has abolished the Ca2+ uptake and ATPase activity of sarcoplasmic reticulum isolated from fast twitch and cardiac muscle but had no influence on either the plasma membrane Ca2+ATPase or Na,K ATPase (Lytton, J. et a/., 1991. Thaps/garg/n inhibits the sarcoplasmic or endoplasmic reticulum Ca2+ ATPase family of calcium pumps. J.Biol.Chem 266(26): 17067-17071). 2,5-Di (tert-butyl)-1,4-benzohydroquinone (BHQ) has been shown to inhibit he Ca2+, Mg2+ -ATPase of sarcoplasmic reticulum with an affinity of 0.4uM (Wictome.M. et a/.,1992. The inhibitors thapsigargin and 2,5 di (ter-butyl)-1,4-benzohydroquinone favour the E2 form of Ca2* Mg2+ ATPase. FEBSLetts.304: 109-113). Bis, (2hydroxy-3-tert-5-methyl phenyl) methane bis-phenol) is the most potent inhibitor of the Ca2+ Mg2+ ATPase of skeletal muscle sarcoplasmic reticulum identified (Sokolove.P.M. et a/., 1986. Phenolic antioxidants.potent inhibitors of the Ca2+ Mg2* ATPase of sarcoplasmic reticulum. FEBSLetts. 203: 121-126). Calponin is a thin filament associated protein that has

been implicated in the regulation of smooth muscle contraction since the isolated protein inhibits the actin activated Mg2+ATPase activity of phosphorylated smooth muscle myosin (Winder and Walsh, 1990. Smooth muscle calponin inhibition of actomyosin Mo2* ATPase and regulation by phosphorylation. J. Biol. Chem 265: 10148-10155).
Reference may be made to Martnez-azorin.F., et a/., 1992. {Effect of diethylstilbestrol and related compound on the Ca2+ transporting ATPase of sarcoplasmic reticulum. J.Biol.Chem. 267(17): 11923-11929) where described about diethylstilbestrol, is a potent inhibitory agent of the Ca2+ ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects.
Reference may be made to Highsmith et a/., 1998 {Reversible inactivation of myosin subfragement 1 activity by mechanical immobilization. Biophys.J. 74(3): 1465-1472) showed that addition of a high molecular weight co-solvent, polyethylene glycol caused aggregation. This aggregation is reported by the addition of 20% PEG which accompanied by the reversible loss of the Mg2+ mediated ATPase activity when measured in low ionic strength. S1 aggregation induced by PEG restrains mechanically the lever arm movement from the closed to open position and because of tight coupling this prevents the M**ADP.Pi to M*ADP transition and inhibits the Mg2+ATPase activity of S1 (Peyser et a/., 2003. Cosolvent induced aggregation inhibits myosin ATPase activity by stabilizing the predominant transition intermediate. Biochemistry 42: 12669-12675). Polyethylene glycol decreased the ATPase activity by 25-40% (Chinn et a/., 2000, Effect of polyethylene glycol on the mechanical and ATPase activity of active muscle fibres. Biophys.J. 78(2): 927-939).
Reference may be made to US patent no.6495337 .Hartman et a/., Dec17 2002 High thorouput sarcomeric assay where describes about the calcium requirement of ATPase of sarcomere.Reference may be made to US patent no.6509167, Hartman et al., Jan 21 2003 High thorouput sarcomeric assay where describes about the ATPase of cardiac sarcomere.
Reference may be made to US patent no.6573061 ,Hartman et al., June 3 2003 sarcomere protein composition where describes about the compounds that modulates the biological activity of biochemically functional sarcomere. Objectives of invention
The main objective is to provide a process for inhibiting the adenosinetriphosphatase(ATPase) activity of fish actomyosin using ployhydric alcohols. Summary of invention
Accordingly, the present invention provides a process for inhibiting the adenosinetriphosphatase(ATPase) activity of fish actomyosin using polyhydric alcohols comprising;
a) isolating actomyosin from fish which is used in Indian oil sardine {Sardinella longiceps) actomyosin;
b) quantifying the enzyme activity of actomyosin;
c) characterized in that estimating the ATPase activity of actomyosin in presence of selected polyhydric alcohol used is sorbitol in the concentration of 6-10% (w/v) and mannitol in the concentration range 4-8% (w/v).
d) elucidating the inhibitory effect of above compounds in pH in the range of 7-9 and at temperature ranging 25°C- 45°C
In an embodiment of the process the polyhydric alcohol used is sorbitol in the concentration of 6-10% (w/v) and mannitol in the concentration range 4-8% (w/v).
In an another embodiment of the process the the fish actomyosin used is Indian oil sardine (Sardinella longiceps) actomyosin.
Detailed description of invention
Actomyosin was isolated from fresh Indian oil sardine {Sardinella longiceps) and was tested for its Ca2+ATPase activity according to the method of Perry, 1955 (Myosin adenosinetriphosphatase, Methods in enzymology, 2, 582). The substrate contains 0.05ml of 50mM ATP, 0.2ml of 0.1 M CaCI2 and in presence of different concentrations of selected ployhydric alcohols. Activity of the enzyme was assayed in presence of different concentrations of sorbitol and mannitol in the substrate. The activity was continued for 15 minutes in a temperature controlled shaking water bath. The reaction was terminated by addition of 1ml 15% trichloroacetic acid. The amount of inorganic phosphate in the supernatant was determined according to Taussky and Shorr, 1953 {Microcalorimetric method for determination of inorganic phosphorous, J Biol Chem, 202:675). The activity of enzyme at 25°C is taken as 100% and the activity of enzyme in presence of different concentrations of polyhydric alcohols and temperatures were expressed as percentage based on this activity.
Examplei
In order to find out the effect of sorbitol and mannitol on Ca2+ATPase activity of actomyosin at pH 7.0 and temperature 25°C the activity was carried out in presence of Tris-CI buffer containing 0.5M KCI in presence of different concentrations of sorbitol or mannitol- control, 2%, 4%, 6%, 8% and 10% in the reaction mixture.
Table 1. Percentage activity of Ca2+ATPase of actomyosin from fish in presence of polyhydric alcohols at pH 7.0 and temperature 25°C

concentrations control 2% 4% 6% 8% 10%
Sorbitol 100 92.5 22.4 5.1 1.7 0
Mannitol 100 92.8 4.4 0 0 0

Example 2
In order to find out the effect of sorbitol and mannitol on Ca2+ATPase activity of actomyosin at pH 7.0 and temperature 37°C the activity was carried out in presence of Tris-CI buffer containing 0.5M KCI in presence of different concentrations of sorbitol or mannitol- control, 2%, 4%, 6%, 8% and 10% in the reaction mixture.
Table 2. Percentage activity of Ca2*ATPase of actomyosin from fish in presence of polyhydric alcohols at pH 7.0 and temperature 37°C

concentration control 2% 4% 6% 8% 10%
Sorbitol 40.5 41.6 12.0 5.1 5.1 0
Mannitol 39.8 15.5 0 0 0 0
Example 3
In order to find out the effect of sorbitol and mannitol on Ca2+ATPase activity of actomyosin at pH 7.0 and temperature 458C the activity was carried out in presence of Tris-CI buffer containing 0.5M KCI in presence of different concentrations of sorbitol or mannitol - control, 2%, 4%, 6%, 8% and 10% in the reaction mixture.
Table 3. Percentage activity of Ca2+ATPase of actomyosin from fish in presence of polyhydric alcohols at pH 7.0 and temperature 45°C

concentration control 2% 4% 6% 8% 10%
Sorbitol 5.2 4.6 1.5 0 0 0
Mannitol 5.9 4.4 0 0 0 0
Example 4
In order to find out the effect of sorbitol and mannitol on Ca2+ATPase activity of actomyosin at pH 9.0 and temperature 25°C the activity was carried out in presence of Glycine -NaOH buffer containing 0.5M KCI in presence of different
8

concentrations of sorbitol or mannitol - control, 2%, 4%, 6%, 8% and 10% in the reaction mixture.
Table 4. Percentage activity of Ca2+ATPase of actomyosin from fish in presence of polyhydric alcohols at pH 9.0 and temperature 25°C

concentration control 2% 4% 6% 8% 10%
Sorbitol 100 88.7 62.5 7.4 0 0
Mannitol 100 74.6 4.4 0 0 0
Example 5
In order to find out the effect of sorbitol and mannitol on Ca2+ATPase activity of actomyosin at pH 9.0 and temperature 37°C the activity was carried out in presence of Glycine -NaOH buffer containing 0.5M KCI in presence of different concentrations of sorbitol or mannitol- control, 2%, 4%, 6%, 8% and 10% in the reaction mixture.
Table S. Percentage activity of Ca2+ATPase of actomyosin from fish in presence of polyhydric alcohols at pH 9.0 and temperature 37°C

concentration control 2% 4% 6% 8% 10%
Sorbitol 22.4 20.6 17.6 5.2 1.1 0
Mannitol 22.1 19.6 1.1 0.5 0 0
Example 6
In order to find out the effect of sorbitol and mannitol on Ca2+ATPase activity of actomyosin at pH 9.0 and temperature 45°C the activity was carried out in presence of Glycine -NaOH buffer containing 0.5M KCI in presence of different concentrations of sorbitol or mannitol, control, 2%, 4%, 6%, 8% and 10% in the reaction mixture.

Table 6. Percentage activity of Ca2+ATPase of actomyosin from fish in presence of polyhydric alcohols at pH 9.0 and temperature 45°C

concentration control 2% 4% 6% 8% 10%
Sorbitol 3.9 3.9 0 0 0 0
Mannitol 3.2 2.5 0 0 0 0












We claim
1. A process for inhibiting the adenosinetriphosphatase(ATPase)
activity of fish actomyosin using polyhydric alcohols comprising;
a) isolating actomyosin from fish which is used in Indian oil sardine (Sardinella longiceps) actomyosin;
b) quantifying the enzyme activity of actomyosin;
c) characterized in that estimating the ATPase activity of actomyosin
in presence of selected polyhydric alcohol used is sorbitol in the
concentration of 6-10% (w/v) and mannitol in the concentration
range 4-8% (w/v).
d) elucidating the inhibitory effect of above compounds in pH in the
range of 7-9 and at temperature ranging 25°C- 45°C
2. A process for inhibiting the adenosinetriphosphatase(ATPase)
activity of fish actomyosin using polyhydric alcohols substantially
as herein described with reference to the examples accompanying
this specification.












Documents:

550-DEL-2004-Abstract-(22-06-2010).pdf

550-del-2004-abstract.pdf

550-DEL-2004-Claims-(22-06-2010).pdf

550-del-2004-claims.pdf

550-DEL-2004-Correspondence-Others-(22-06-2010).pdf

550-del-2004-correspondence.pdf

550-DEL-2004-Description (Complete)-(22-06-2010).pdf

550-del-2004-description.pdf

550-DEL-2004-Form-1-(22-06-2010).pdf

550-DEL-2004-Form-3-(22-06-2010).pdf

550-del-2004-form1.pdf

550-del-2004-form2.pdf

550-del-2004-form3.pdf

550-del-2004-form5.pdf


Patent Number 243021
Indian Patent Application Number 550/DEL/2004
PG Journal Number 40/2010
Publication Date 01-Oct-2010
Grant Date 23-Sep-2010
Date of Filing 22-Mar-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 SIJO MATHEW CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
2 VISHWESHWARAIAH PRAKASH CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
PCT International Classification Number C 07 K 14/46
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA