Buffalo Kidney L-Gulonate Dehydrogenase: Isolation And .

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World J Life Sci. and Medical Research 2013;33(1):8Sharma et al., 2013.2013 Buffalo Kidney L-Gulonate ISSN 2249-0574Buffalo Kidney L-GulonateDehydrogenase: Isolation and KineticCharacterizationVineet SHARMA 1, Shah Saddad HUSSAIN 1, Shrawat POOJA 2, Jangra SONIA 2,K NIRMALA 2, Kambadur MURALIDHAR 1ABSTRACT ons:L-Gulonate dehydrogenase (L-GuDH) from buffalo kidney has been investigated with regard to thefeasibility of purification and some kinetic properties. Salt fractionation with ammonium sulfate, SPSephadex chromatography, Hydroxyapatite chromatography, among the resins tested, have shownpromise for enrichment of this enzyme. Pseudoaffinity chromatography on immobilized Cibacron BlueF3GA was not useful for enrichment of L-GuDH from buffalo kidney. L-Gulono-γ-lactone and L-Ascorbicacid were found to inhibit the enzyme activity. The physiological significance of these observations todiabetic nephropathy has been discussed.1Hormone Research Laboratory,Department of Zoology, Daulat RamCollege, University of Delhi, DELHI2Department of Biochemistry,Daulat Ram College, University ofDelhi, DELHI* Email Address forCorrespondence/ Adresse decourriel pour la correspondance:kambadur@hotmail.comKeywords: Buffalo kidney, Gulonate dehydrogenase, Gulono lactoneRÉSUMÉ [FRANÇAIS/FRENCH]FRANÇAIS/FRENCH]L-gulonate déshydrogénase (L-GuDH) à partir de rein de buffle a été étudiée en ce qui concerne lafaisabilité de purification et de certaines propriétés cinétiques. Fractionnement de sel avec du sulfated'ammonium, SP-Sephadex, Chromatographie d'hydroxyapatite, parmi les résines testées ont montré desrésultats prometteurs pour l'enrichissement de cette enzyme. Pseudoaffinity chromatographie surimmobilisée F3GA Cibacron Blue n'était pas utile pour l'enrichissement de la L-GuDH de rein de buffle. Lgulono-γ-lactone et d'acide L-ascorbique ont été trouvés pour inhiber l'activité de l'enzyme. Lasignification physiologique de ces observations pour la néphropathie diabétique a été discutée.Mots-clés: Rénale Buffalo, gulonate déshydrogénase, gulono lactoneAccepted/Accepté: February, 2013Full Citation: Sharma V, Hussain SS,Pooja, Sonia, Nirmala K, MuraliharK. Buffalo Kidney L-GulonateDehydrogenase: isolation and kineticcharacterization. World Journal LifeScience and Medical Research.2013;3(1):8-14.INTRODUCTIONascorbic acid formation, owing to increase in gulonateL-Gulonate dehydrogenase (EC 1.1.1.45) is a key enzymedehydrogenase activity [3]. Hence to understand theof the pentose phosphate cycle. The substrate of thisphysiologicalenzyme i.e. L-Gulonic acid occupies a unique position in aregulation, purification and characterization of GuDHmetabolic traffic junction. L-Gulonic acid hypotheticallyfrom buffalo kidney has been attempted by investigatinghas equal choice to enter into either the pentose phosphateits interaction with different chromatographic matrices.significanceofthismetabolitefluxmetabolic pathway to be metabolized into L-xylitol or IALS AND METHODSconsidered one of the water-soluble vitamins required forL-Ascorbic acid, β-hydroxy butyrate and L-Gulonic acid-the successful completion of the reproductive processes inƳ-lactone were purchased from Sigma Aldrich, USA.majority of mammals. It is found stored in endocrineNAD and NADH were procured from Spectrochem.tissues like adrenals and in reproductive tissues [1)attesting to its possible metabolic role in gonadalPreparation of LL-Gulonic Acidphysiology [2]. Biosynthesis of ascorbic acid seems to be200 mM L-gulonic acid-Ƴ-lactone was made in 50 mMdirectly related to the expression of the L- Gulonatephosphate buffer, pH 7.5. The pH of the solution wasdydrogenase. During starvation, the metabolite flux isbrought to 12.0 by adding 1M NaOH drop wise and leftfound shifted towards L-xylulose formation, rather thanfor one hour with constant stirring. Finally, the pH of theOPENACCESS Research Reviews Publications, 2013http://www.rrpjournals.com/

ISSN 2249-0574World J Life Sci. and Medical Research 2013;33(1):9al.,., 2013.Sharma et al2013 Buffalo Kidney L-Gulonate DehydrogenaseOPENACCESSsolution was brought back to 7.5 by adding 1N HCl droppH 7.5 and bound proteins were eluted with increasingwise. This solution is used as the enzyme substrate in thediscontinuous concentration of phosphate buffer (20-200enzyme assays.mM).Gulonate dehydrogenase was also loaded on Phenyl-Interaction of LL-Gulonate Dehydrogenase fromBuffalo Kidney with Various Matricesbuffer, pH 7.5, 1M NaCl) and later bound proteins wereBuffalo kidney was brought from the Gazipur abattoireluted with decreasing concentration of salt and ethylene(Delhi government unit near Noida) to our laboratory inglycol.an ice box. The adhering membranous covering wasTheremoved. The cortical portion was cut into small piecesimmobilized metal affinity chromatography (IMAC) byand later homogenized in 50 mM PB, pH 7.5 containing 1using different metal ions namely nickel and cobalt. IDA-mM PMSF (Buffer A) using a kitchen blender. Finally theSepharose matrices were recharged with 1M metal salthomogenate was centrifuged at 20,000 g for 30 min at 4oC.solutionsThe clear supernatant was termed as “crude extract”equilibrated with 20 mM phosphate buffer, pH 7.5; 0.5 Mwhich was ultimately subjected to ammonium sulfateNaCl and enzyme preparation were loaded at the samesepharose at high salt concentration (50 mM Cl2.6H20).loadedColumnsonwereprecipitation (0-30% and 30-60% saturation). The pelletspH on the column. After removing unbound fractions,recovered from various steps were assessed for enzymebound proteins were eluted out by 300 mM purepreparation was finally used for the investigation of theBiochemical and Zymogram Assays of LL-GuDHinteraction of buffalo kidney L-Gulonate dehydrogenaseEnzyme activity of Gulonate dehydrogenase was assayedwith various types of chromatographic matrices includingby measuring the rate of change in NADH absorbance atHydroxyapatite, Blue-sepharose, Yellow-Sepharose, SP-340 nm. Km and Vmax was calculated by keepingSepharose,increasing Gulonate concentration between 1-40 mM [4].CM-sepharose,DEAE-Sephadex,Phenyl-Sepharose, and IMAC matrices.Enzyme inhibition studies were also performed byIn dye affinity chromatography (Blue-Sepharose epharose), the protein solution was loaded on theconcentration and Ki was calculated from the graphBlue-Sepharose/Yellow-Sepharose at pH 5.0 and boundbetween KM apparent Vs inhibitor concentration and from theproteins were eluted with 50 mM citrate buffer, pH 5.0, 50graph between KMapparent Vs inhibitor concentration. ThemM phosphate buffer,in-gel enzyme activity staining method was also used topH 6.0, 50 mM phosphate buffer, pH 7.5 and 50 mMdetectphosphate buffer, pH 7.5 containing 1 M NaCl. The sameconcentration was determined by Lowry’s method usingpreparation was also loaded at pH 7.5 and bound proteinsBSA as a standard [6].enzymeactivityqualitatively[5].Proteinwere eluted with 50 mM phosphate buffer, pH 7.5 and 50mM phosphate buffer, pH 7.5 containing 1M NaCl.RESULTS AND DISCUSSIONSemi-pure preparation of the Gulonate dehydrogenaseL-Gulonate dehydrogenase (L-GuDH) is a physiologicallywas also loaded on various ion exchangers. It was loadedimportantonto(SP-Sephadex/CM-dehydrogenation of L-Gulonate into dehydro-L-Gulonatecellulose) which were previously equilibrated with 50 mMin the uronate cycle [7]. Regarding this enzyme, particularcitrate buffer (pH 5) having 50 mM NaCl. Bound proteinsareas of interest include details of substrate catalysis,were eluted with 50 mM phosphate buffer, pH 6, 50 mMactive site modification, substrate-binding-site specificity,phosphate buffer, pH 7.5 and 50 mM phosphate buffer,kinetics, and stability. To get more understanding of itspH 7.5 having 500 mM NaCl. Gulonate dehydrogenasestructural features, interaction behavior of buffalo kidneywas also loaded on anion exchanger like DEAE-SephadexL-Gulonateat pH 7.5, equilibrated with 50 mM phosphate buffer (pHchromatographic matrices have been attempted. This can7.5) having 50 mM NaCl. The bound proteins were elutedlead to the formulation of a purification procedure for thewith discontinuous gradient of salt (50 mM-1M NaCl).Gulonate dehydrogenase.Hydroxyapatite chromatography was also used to purifyBuffalo kidney L-Gulonate dehydrogenase was subjectedGulonate dehydrogenase from buffalo kidney. Theto ammonium sulfate precipitation and was found toenzyme extract was loaded at 20 mM phosphate buffer,concentrate mostly in the 30%-60% ammonium ydrogenasethewith Research Reviews Publications, 2013http://www.rrpjournals.com/NAD -linkeddifferentOPENACCESS

World J Life Sci. and Medical Research 2013;33(1):10Sharma et al., 2013.2013 Buffalo Kidney L-Gulonate DehydrogenaseOPENACCESSpellet ( 82% of the starting enzyme units) fraction.ISSN ferent fractions were assayed for the enzyme activityconcentrate enzyme activity in unbound fractions andby using classical spectroscopic dehydrogenase assay.bound fractions were found to be devoid of any enzymeSpecific activity of the final preparation was found to beactivity (Fig 1). Buffalo kidney extract has been loaded on0.002051Gulonateto SP-Sephadex and Gulonate dehydrogenase activity hasdehydrogenase has been found to be purified by 3.53 foldbeen found to be solely restricted to unbound fractionsin comparison to crude extract, while very little enzyme(Fig 2 and Table 2). Interestingly Gulonate dehydrogenaseU/mgwith82.71%yield.activity has been observed in other side fractions of salthas been found to bind CM- Sephadex and elute at pH 7.5fractionation step (Table 1). One unit of the enzyme was(Fig 3). Presence of the enzyme activity in unbounddefined as the amount of the enzyme required to reducefractions can be ascribed as overloading of the protein on1µmol of NAD.the column (Table 3).Table 1: This table shows summary of the partial purification of the Gulonate dehydrogenase from buffalo kidneyStepsTotal Volume(mL)1Crude10090007.04250.0007831100230 P3.4214.20.12450.0005810.7427891.77360 P4028405.8250.0020513.52880582.71460 S6.8170.000000Figure1:ThisfigureTotal Protein(mg)showsTotal Activity(µmoles/min)Specific hydroxyapatiteeluted with high salt concentration suggesting presence ofchromatography elution profile of buffalo kidney extractthe less positive charge on Gulonate dehydrogenase at pHfor the interaction studies of buffalo kidney L-Gulonate7.5. (Fig 4; Table 4).dehydrogenase with romatography elution profile of buffalo extracts for drogenase with SP-Sephadex.Hydroxyapatite chromatography has been reported as enase (K. Muralidhar- personal communication).When kidney extract was subjected to anion exchanger(DEAE-Sephadex), bound Gulonate dehydrogenase wasIn dye affinity chromatography, protein binding wasfound to be eluted in two fractions, mostly with buffer 2found to be dependent on the initial loading pH. At pH(50 mM PB, pH 7.5, 100 mM NaCl), while another isoform5.0 Gulonate dehydrogenase seems to bind the Blue-eluted later with buffer 3 (50 mM PB, pH 7.5, 200 mMSepharose, while at pH 7.5 almost nothing bound (dataNaCl). No enzyme activity appeared in the fractionsnot shown). Bound Gulonate dehydrogenase eluted withOPENACCESS Research Reviews Publications, 2013http://www.rrpjournals.com/

ISSN 2249-0574World J Life Sci. and Medical Research 2013;33(1):11al.,., 2013.Sharma et al2013 Buffalo Kidney L-Gulonate DehydrogenaseOPENACCESSphosphate buffer pH 7.5 but there was not much increaseIonic interaction seems to dominate these bindingin specific activity. Interestingly contrast observationsinteractions.were reported with regards to binding behavior of L-observed when Gulonate dehydrogenase was subjected toGulonate dehydrogenase towards Blue-Sepharose [7].reactiveTable othasbeenshown).This table shows summary of the purification profile of the Gulonate dehydrogenase from buffalo kidney byusing SP-Sephadex Buffer 1: 50 mM citrate buffer pH 5 50 mM NaCl ; Buffer 2: 50 mM phosphate buffer pH 6; Buffer 3:50 mM phosphate buffer pH 7.5 and Buffer 4: 50 mM phosphate buffer pH 7.5 500 mM NaClSno.StepsVolume(ml)Total Protein(mg)Total Activity(umoles/min)Specific .056340.00031312Buffer 1 (F1)24.320.0079180.0018335.8558213Buffer 1 (F2)230.0090840.0030289.6736974Buffer 2 (F1)20.30005Buffer 3 (F2)230.000040.0000130.0415346Buffer 4 (F2)20.3000Table 3:This table shows summary of the purification profile of the Gulonate dehydrogenase from buffalo kidney byusing CM-Sephadex. (Buffers 1-4 mean the same as in Table 2).S/NoSampleVolume(ml)Total Protein(mg)Total Activity(umoles/min)Specific .056340.00031312Buffer 1 (F1)21.660.0025320.0015254.8734643Buffer 1 (F2)20.660.0012060.0018275.8369054Buffer 2 (F1)20.30.000040.0001330.4259855Buffer 3 (F1)20.50.0009240.0018495.9069486Buffer 3 (F2)24.320.0012860.0002980.9511997Buffer 4 (F2)20.440.000080.0001820.580889Table 4:This table shows summary of the purification profile of the Gulonate dehydrogenase from buffalo kidney byusing DEAE-Sephadex. (Buffers mean the same as in Table 2).S/NoSampleVolume(ml)Total Protein(mg)Total Activity(umoles/min)Specific .056340.00031312Buffer 1 (F1)211.20.0005630.000050.1597443Buffer 1 (F2)290.0028140.0003126120.9987594Buffer 1 (F3)250.0032960.0006591642.1059555Buffer 2 (F3)20.540.0030950.00573121418.310596Buffer 2 (F4)20.760.0023710.003120249.9688197Buffer 3 (F3)20.90.0008840.0009824943.1389588Buffer 3 (F4)20.760.0006030.0007932812.5344459Buffer 4 (F2)2100010Buffer 5 (F2)20.44000 Research Reviews Publications, 2013http://www.rrpjournals.com/OPENACCESS

World J Life Sci. and Medical Research 2013;33(1):12Sharma et al., 2013.2013 Buffalo Kidney L-Gulonate DehydrogenaseOPENACCESSISSN 2249-0574Semi-pure preparation was loaded on immobilized metalenzyme activity was also retained in bound fractions.affinity chromatography (IMAC) by using different metal36.62% of the total proteins were found to interact withions namely nickel and cobalt. Both metal ions wereNi-IDA sepharose (9.4 X 1018 molecules Ni2 /mL matrix).found to bind protein significantly, while significantCobalt-Sepharose was shown to interact differently inenzyme activity eluted in unbound fractions. Nickel-comparison to nickel. As per protein absorbance wholeSepharose was shown to bind certain proteins (Fig 4).protein came out as unbound fractions (data not shown).Figure3:ThisfigureshowsCM-SephadexFigure 5:This figure showsNickel-IDA Sepharosechromatography elution profile of buffalo kidney extractchromatography elution profile of buffalo kidney extractfor the interaction studies of buffalo kidney L-Gulonatefor the interaction studies of buffalo kidney L-Gulonatedehydrogenase with CM-Sephadex.dehydrogenase with Ni-IDA pharoseDEAE-Sephadexchromatography elution profile of buffalo kidney extractschromatography elution profile of buffalo extracts for thefor the interaction studies of buffalo kidney L-Gulonateinteractiondehydrogenase with neyL-Gulonatedehydrogenase with DEAE-Sephadex.The cobalt ions bound to IDA matrix were found to be 5.4As per zymogram studies most of the enzyme activityX 1019 Co2 ions/ mL of matrix. Absence of any enzymewas retained in the unbound fractions while someactivity in bound fraction could be explained in terms ofOPENACCESS Research Reviews Publications, 2013http://www.rrpjournals.com/

ISSN 2249-0574World J Life Sci. and Medical Research 2013;33(1):13al.,., 2013.Sharma et al2013 Buffalo Kidney L-Gulonate DehydrogenaseOPENACCESSloss of enzyme activity after the interaction of metals withDifferent biomolecules with similar structure to L-gulonicthis enzyme as many ions have been shown to affect theacid can hypothetically be inhibitors of this enzyme.enzyme activity of L-GuDH previously [7]. PhenylMetabolic controlSepharose is a separation medium for hydrophobicenzyme expression in vivo can be achieved by ascorbicinteractionacid or L-gulono-Ƴ-lactone. The kinetic parameter dehydrogenaseseparated on the basis of their different hydrophobicity.Km was found to be 2 to 7 mM by different reciprocalGulonate dehydrogenase has been found to bind Phenyl-plots (Table 6). L-Ascorbic acid and L-Gulono–γ-lactoneSepharose at high salt concentration and two isoformswere tested as potential inhibitors. Indeed resultswere eluted with different eluting buffers. One activityindicated that both could inhibit the enzyme activity in apeak eluted with phosphate buffer without any salt whilecompetitivemode.another highly hydrophobic has been eluted withethylene glycol (Fig 6 and Table 5).Table 5:This table shows summary of the purification profile of the Gulonate dehydrogenase from buffalo kidney byusing Phenyl-Sepharose.S/NoStepsVolume(ml)Total Protein(mg)Total Activity(umoles/min)Specific .056340.00031312Buffer 1 (F1)20.660.0001210.0001830.583693Buffer 1 (F2)20.160004Buffer 2 (F2)20.660.0175240.02655284.829685Buffer 3 (F2)23.460.00410.0011853.785553Figure 7:This figure shows kinetics of GuDH inhibition by L-gulono-γ-lactone. GuDH was incubated with increasingconcentration of gulonic acid at different concentration of inhibitor. Secondary replots of the double reciprocal plots, i.e.,KM (B) and slope(C) Vs inhibitor concentrations.Experimentally determined Ki values obtained fromand Phenyl-Sepharose chromatography. Dye affinityreciprocal plots were found to be 5.332 mM and 4.17 mMchromatography has been not useful for the purificationfor the L- gulono-γ-lactone (Fig 7). The values were 2.83of this enzyme. Metal ions were shown to interactmM and 6.92 mM for ascorbic acid (data not shown). Indifferently with the proteins present in the semi-puresummary, purification of Gulonate dehydrogenase can bepreparation. Many of the matrices have shown theachieved by coupling salt fractionation, Hydroxyapatitepotentialofusingthemasapurification Research Reviews Publications, 2013http://www.rrpjournals.com/andOPENACCESS

World J Life Sci. and Medical Research 2013;33(1):14Sharma et al., 2013.2013 Buffalo Kidney L-Gulonate DehydrogenaseOPENACCESScharacterizationstep ofL-Gulonatedehydrogenase.regulatoryInhibition studies of Gulonate dehydrogenase by ascorbicISSN 2249-0574mechanismofGulonatedehydrogenaseactivity under in vivo conditions.acid and L-gulono-γ-lactone shed light on the lonatedehydrogenasededucedfromdifferent reciprocal plots.S/NoKinetic plotsKM (mM) Vmax (µmole Hanes-Woolf2.2020.002179CONCLUSIONbuffalo kidney by naturally occurring Metabolites.It is concluded that different chromatography resins were.Novus Int J Pharmaceut.l Technol 2012;1(4):19-29.[5]tried to know whether L-GuDH from buffalo kidneySharma V, Chaudhary R, Khurana JM, Muralidharcould be enriched in specific activity. Hydroxyapatite andK.Phenyl-Sepharose were useful among these resins.nitrprusside-thiolImmobilized textile dye (i.e. Cibacron Blue F3GA) was notAnal.2008;19:99-103.[6]found useful for this tochem.Lowry OM, Rosenbrough NJ, Farr AJ, Randall RJ.Protein measurement with the Folin phenol reagent,REFERENCES[1][2]J. Biol. Chem.1951;193:265-75.Hurley WL, Doane RM. Recent developments in the[7]Ishikura S, Usami N, Araki M, Hara A. Structuralroles of vitamins and minerals in reproduction. J.and Functional Characterization of Rabbit andDairy Sci.,1989;72(3):784- 804.Human L-Gulonate 3-Dehydrogenase, J. Biochem.Luck MR, Jeyaseelan I, Scholes RA. Ascorbic Acid2005,137,303-314.and Fertility Biol. Reproduc.,1995;52:262-6.[3]Dykhuizen DE, Harrison KM, Richardson BJ.ACKNOWLEDGEMENTACKNOWLEDGEMENT / SOURCE(S)SOURCE(S) OF SUPPORTEvolutionary implicationsNilof ascorbicacidproduction in the Australian lungfish ExperientiaCONFLICT OF INTEREST1980;36:945-6.[4]Hussain SS, Sharma M, Nangia M, Muralidhar, K.InhibitionofL-GulonateNildehydrogenase fromHow to Submit ManuscriptsSince we use very fast review system, and since we are dedicated to publishing submitted articles with few weeks ofsubmission, then the easiest and most reliable way of submitting a manuscript for publication in any of the journalsfrom the publisher Research, Reviews and Publications (also known as Research Reviews Publications) is bysending an electronic copy of the well formatted manuscript as an email attachment to rrpjournals@gmail.com oronline at http://www.rrpjournals.com/.Submissions are often acknowledged within 6 to 24 hours of submission and the review process normally starts withinfew hours later, except in the rear cases where we are unable to find the appropriate reviewer on time.Manuscripts are hardly rejected without first sending them for review, except in the cases where the manuscripts arepoorly formatted and the author(s) have not followed the instructions for manuscript preparation which is InstructionsForAuthors.html .Research Reviews Publications and its journals have so many unique features such as rapid and quality publication ofexcellent articles, bilingual publication, some of which are available at http://www.rrpjournals.com/uniqueness.html .OPENACCESS Research Reviews Publications, 2013http://www.rrpjournals.com/

immobilisée F3GA Cibacron Blue n'était pas utile pour l'enrichissement de la L-GuDH de rein de buffle. L-gulono-γ-lactone et d'acide L-ascorbique ont été trouvés pour inhiber l'activité de l'enzyme. La signification physiologique de ces obs

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