Compared to the other proteins in the rat kidney, what characteristics (quantity and charge) of the sphingosine kinase enzyme can you infer from its elution behavior from the DEAE column? Utilize specific observations from the graph in your answer to support your statements. The investigators purified the enzyme sphingosine kinase using rat kidneys as asource of enzyme since it had been shown previously that kidneys contain a highconcentration of SPP. Kidneys were homogenized in buffer, filtered, thenfractionated by ammonium sulfate precipitation. The ammonium sulfate precipitatewas dissolved in buffer and then applied to a DEAE-cellulose (anion exchange)column.The investigators eluted the protein using Tris buffer at pH 7.4 with increasingconcentrations of sodium chloride (from 0.00 to 0.50 M). Fractions were collectedand assayed for sphingosine kinase in a procedure that involved the addition ofsphingosine to radioactively-labeled y³²P-ATP (that is, the y phosphate group on theATP contains the isotope 32P) and the subsequent quantitation of 32P in the SPPproduct. The results are shown in the figure below.Kinase activity, units20000150001351910000500000it bonisido tolqOM Nad50100el bas der0.5 M Nad80 15025Fraction number-- Kinase activity, units - Protein, mgFUide 20sahiptonigh151000000-005200Protein, mgThe dotted line represents the sodium chloride concentration of the eluting fluid. Task 12: Case Study: Purification of Rat Kidney Sphingosine KinaseObjective: Becoming familiar with the purification and kinetic analysis of an enzymethat produces a product important in cell survival.Introduction: Sphingolipid metabolites have recently been shown to be involved incell viability. For example, ceramide has been shown to stimulate apoptosis, orprogrammed cell death, whereas sphingosine-1-phosphate (SPP) promotes cellsurvival. A number of different factors influence the levels of ceramide and SPP inthe cell, as shown in the figure below.Anticancer drugsomme d Platelet-derived growth factor fud ni boxingRadiation Ads of boil TNF-a.Other growth factorsmotor (4 02.0 or 0 m)oldovode sus eluen oi, Joobong 792 odi ni 9° to noitsinsup roupsedre or box ITA (CeramideSpingosine-1-phosphate (SPP)HO HOProgrammed Cell Death(Apoptosis)od orthatnomstura tardw.yombil temat ni anistong modto odi of borsali no board omysho sanomzognirige ont mode solem inYanolo BANC or moft toivadod aClearly, the balance of ceramide and SPP will be one of the many factors thatinfluences whether a cell lives or dies. An understanding of the biochemicalprocesses involved in regulating these sphingolipids has important applications inthe treatment of cancer.HOSPP is synthesized in the cell from sphingosine and ATP as shown in the figurebelow. The enzyme that catalyzes the reaction is sphingosine kinase. We will studythe purification of this enzyme.Cell Survivalcogaidoe sdi indi bahoqui anotagiteovsono 2000Invivius lloo ni ushoqni bouborga asouborg tadt omvano na to elevisce oitonibl bas noitsofting of-Obute aid to anoot odO=POHCH₂-CH- -CHOон aiuротот?abodtom ispitylaus nistong bra asupurtost nollsoft istoriCH₂-CH-CHEsphingosinekinaseNH₂* CH+ ATPNH₂CHA ADPolgmaxs 104 yily liso mi bovlovni od of wors nood vilnoot ovad estilodator-1-onizognidge as CHw disob lleo berunsigong 10 juizolqoqs sisturcite of awode CHobiumino to sloval odconsufint exotont moroftib to todmun A devive liso estomong (998) oledgeadq(CH₂)121.01 sugil of awora as (CH₂)12 992 bastodiarw zoontlini soli exoral vason si to ono od lliw 198 ban obimsiso to sonsindo virolobasit guitsli CH₂i bovlovni 2082soong isoimodooid oda to guibretersbou nA CH₂ 10 eovil leo-oxisognidas no slitsono liw Woonso to Inomtead siit al anoitepilqgs Instrogmi ead abigilogmulga

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Formation of the peptide bond neutralises any charge due carboxyl/amino group, therefore the net…