full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1196divided into two parts. One part was centrifugedat 1400g <strong>and</strong> the supernatant thus obtained wasused as an enzyme source for Mg 2+ ATPase, whilethe other part <strong>of</strong> the homogenate was used forthe estimation <strong>of</strong> the total ATPase.Mg 2+ ATPase: The reaction mixture forMg 2+ ATPase assay contained 0.5 ml <strong>of</strong> tris buffer(0.13 M; pH 7.4), 0.4 ml <strong>of</strong> substrate ATP, 0.5 ml<strong>of</strong> Magnesium chloride (0.05 M MgCl 2) <strong>and</strong> 0.2ml <strong>of</strong> mitochondrial fraction (enzyme source). Thecontents were incubated at 37° C for 15 minutes<strong>and</strong> the reaction was stopped by the addition <strong>of</strong>10% TCA. Zero time controls were maintainedby adding TCA prior to the addition <strong>of</strong>homogenate/mitochondrial fraction. The contentswere centrifuged at 1000g for 15 minutes <strong>and</strong>the inorganic phosphate was estimated in thesupernatant fraction following the method <strong>of</strong> Fiske<strong>and</strong> Subbarow (20).Na + K + ATPase: 1% (W/V) homogenate alreadyset apart was used for the total ATPase assay.The reaction mixture in a final volume <strong>of</strong> 2.6 mlcontained, 0.5 ml <strong>of</strong> Tris buffer (0.13 M; pH 7.4),0.4 ml <strong>of</strong> substrate ATP, 0.5 ml MgCl 2(0.05 M),0.5 ml potassium chloride (KCl, 0.05 M), 0.5 ml<strong>of</strong> sodium chloride (NaCl, 0.05 M) <strong>and</strong> 0.2 ml <strong>of</strong>crude homogenate/ mitochondrial fraction(enzyme source). The contents were incubatedat 37° C for 15 minutes <strong>and</strong> the reaction wasarrested by the addition <strong>of</strong> 1.5 ml <strong>of</strong> 10% TCAprior to the addition <strong>of</strong> homogenate. The contentswere centrifuged <strong>and</strong> the inorganic phosphate wasestimated in the supernatant fraction (Na +K + ATPase = Total ATPase - Mg 2+ ATPase).Estimation <strong>of</strong> protein content: Protein content<strong>of</strong> the t<strong>issue</strong>s was estimated by the method <strong>of</strong>Lowry et al. (21).Analysis <strong>of</strong> Data: The data was subjected tostatistical analysis (ANOVA <strong>and</strong> t-test asapplicable) using st<strong>and</strong>ard statistical procedures.ResultsOpen field behavior:The results presented inFig.1 show decreased locomotor behavior in allresponses (crossings, rearings, sniffings,groomings) <strong>of</strong> the open-field in Pb+Mn exposedrats compared to controls. The control rats showedhigher locomotor activity (111.6 crossings, 14.2rearings, 19.2 sniffings, 19.2 groomings) comparedto Pb+Mn treated rats (31.7 crossings, 3.1rearings, 3.7 sniffings, 4.3 groomings). However,the addition <strong>of</strong> calcium to Pb+Mn, showed amarginal reversal in the alterations observed inthe locomotor behavior (46.88 crossings, 4.33rearings, 6.77 sniffings, 7.33 groomings) <strong>of</strong>Pb+Mn treated rats.Locomotor activity: The results <strong>of</strong> locomotoractivity (Fig.2) showed a decrease in the totalnumber <strong>of</strong> movements in the Pb+Mn treated ratsthan the control rats. The administration <strong>of</strong>calcium along with the Pb+Mn, showed increasein the locomotory activity than Pb+Mn exposedrats.Fig.1. Open-field behavior <strong>of</strong> control, Pb+Mn exposed<strong>and</strong> Ca 2+ supplemented rats. Rats were exposed to0.2% lead acetate through drinking water <strong>and</strong> Mn (2.5mg/kg) intraperitonially daily for a period <strong>of</strong> 21 days.Calcium (0.02%) was added in 0.2% Pb water. Each barrepresents mean ± SD (n = 6). The values marked withasterisk (*) are significantly different from controls atp