Please paraphrase how both experiments (from fig.2 and 3) are asking differing questions The results with Triton agreed with earlier observations (6, 11,20-22). Regarding Lubrol WX, calnexin had previously beenfound to be partly insoluble (15), consistent with our results. Incontrast, TfR, which in our hands largely resisted extraction withLubrol WX, was reported to be Lubrol-soluble in MDCKcells (15).Therefore, analysis of the protein contents of DRMS fromMDCK cells showed that the detergents differed considerably intheir ability to enrich strongly lipid-associated proteins; i.e., theydiffered in thcir "DRM selectivities."Lipid Analysis of DRMS Prepared with Different Detergents. Toanalyze the lipid content of DRMS, MDCK and Jurkat cells wereused to label either all lipids with 1"C or phospholipids with 32P.Total cell membranes (TCMS) and DRMS were obtaincd byflotation on sucrose step gradients. Lipids were extracted andresolved by TLC (Fig. 3). As observed for the protein content ofDRMS derived from the plasma membrane, the amounts of lipidpresent in the DRMS varied considerably between detergents.To show qualitative changes in the lipid composition of DRMScompared with TCMS, lipids were grouped into sphingolipids,cholesterol, and glycerophospholipids. Sphingolipid/glycero-Compared with TCMS, DRMS from MDCK cells preparedwith Triton and CHAPS showed clear enrichment of sphingo-lipids and cholesterol over glycerophospholipids. The otherdetergents enriched these lipids less strongly. Nevertheless, theircapacity for enrichment correlated with the DRM specificitywith regard to proteins, such that Brij 98 and Brij 96 caused amore pronounced enrichment of sphingolipids and cholesterolthan Tween 20, Brij 58, and Lubrol WX. The same trend wasobserved for Jurkat cells, but notable differences exist. Contraryto MDCK cells, Brij 98 and Brij 58 enriched sphingolipids andcholesterol to a similar extent, whereas Brij 96 did not.The behaviors of Brij 58, Brij 96, and Brij 98 in MDCK andJurkat cells illustrate that the effect of a given detergent may varybetween cell types. Morcover, the enrichment of sphingolipidsand cholesterol was overall less pronounced in Jurkat cells thanin MDCK cells, showing that the lipid composition of DRMSprepared with a particular detergent can be cell type-dependent.Next, we analyzed the fatty acid substitution pattern ofglycerophospholipids in different DRMS. Lipids from the float-ing fractions of MDCK and Jurkat cells were prepared as before,and PC, the major glycerophospholipid, was analyzed by massspectrometry. Seventeen PC species, differing in the total num-cholesterolcholesterolGluc/GalcGlucPEPELacCPC/PVPSSMPC/PIPSSMGbFig. 3. Lipid contents of DRMS from MDCK and Jurkat cells.Lipids from TCMS and DRMS from 14C- or 12p.labeled MDCK andJurkat cells were analyzed by TLC (Upper, 1"C-labeled lipids;12p-labeled lipids). Gluc/GalC/LacC, glucosyl/galactosyl/PEPE-PIPIㅡPSLower,lactosyl ceramide; SM, sphingomyelin; Gbs pentosylglucoside;PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS,phosphatidylserine. Asterisks denote lipids not identified bycomigration with lipid standards.PSPCPCSM<SMMDCK cellsJurkat cellsPNAS I May 13, 2003 | vol. 100 | no. 10 | 5797Schuck et al.Brij 96Brij 98LubrolBrij 58CHAPSritonBrij 96Brij 98LubrolBrij 58TweenTweenTCM topbottomtopbottomtopbottomVIP17/MALPLAPcaveolin-1stomatinYesTIRgp114calnexinrab-5Lubrol WXBrij 96TritonFig. 2. DRM association of marker proteins from MDCK cells. MDCK-PLAP cell homogenate was extracted with 0.5% Lubrol WX, 0.5% Brij 96, or 1% Triton,and floated on linear sucrose gradients. The distribution of marker proteins was analyzed by immunoblotting. The light fractions from the top of the gradientsare on the left, the heavy bottom fractions on the right.96 additionally solubilized gp114, most of TfR, and -50% of Yes(Fig. 2 Center), whereas Triton solubilized rab-5, calnexin, gp114.TfR, and most of Yes (Fig. 2 Right). Hence, DRM association ofthese marker proteins confirms the graded selectivity of thedetergents obscrved by analysis of the plasma membrane DRMprotein content.The results with Triton agreed with earlier observations (6, 11,20-22). Regarding Lubrol WX, calnexin had previously beenfound to be partly insoluble (15), consistent with our results. Incontrast, TfR, which in our hands largely resisted extraction withLubrol WX, was reported to be Lubrol-soluble in MDCKcells (15).Therefore, analysis of the protein contents of DRMS fromMDCK cells showed that the detergents differed considerably intheir ability to enrich strongly lipid-associated proteins; i.e., theydiffered in their "DRM selectivities."phospholipid and cholesterol/glycerophospholipid ratios werecalculated. The fold changes of these ratios relative to the TCMSare depicted in Fig. 4. No significant changes occurred whenhigher detergent concentrations were used or when DRMSwhere prepared by pelleting from isolated membranes instead offlotation from cell homogenate (not shown).Compared with TCMS, DRMS from MDCK cells preparedwith Triton and CHAPS showed clear enrichment of sphingo-lipids and cholesterol over glycerophospholipids. The otherdetergents enriched these lipids less strongly. Nevertheless, theircapacity for enrichment correlated with the DRM specificitywith regard to proteins, such that Brij 98 and Brij 96 caused amore pronounced enrichment of sphingolipids and cholesterolthan Tween 20, Brij 58, and Lubrol WX. The same trend wasobserved for Jurkat cells, but notable differences exist. Contraryto MDCK cells, Brij 98 and Brij 58 enriched sphingolipids andcholesterol to a similar extent, whereas Brij 96 did not.The behaviors of Brij 58, Brij 96, and Brij 98 in MDCK andJurkat cells illustrate that the effect of a given detergent may varybetween cell types. Morcover, the enrichment of sphingolipidsand cholesterol was overall less pronounced in Jurkat cells thanin MDCK cells, showing that the lipid composition of DRMSprepared with a particular detergent can be cell type-dependent.Next, we analyzed the fatty acid substitution pattern ofslyserophospholipids in different DRMS, Lipids from the float-Lipid Analysis of DRMS Prepared with Different Detergents. Toanalyze the lipid content of DRMS, MDCK and Jurkat cells wereused to label cither all lipids with 14C or phospholipids with 32P.Total cell membranes (TCMS) and DRMS were obtained byflotation on sucrose step gradients. Lipids were extracted andresolved by TLC (Fig. 3). As observed for the protein content ofDRMS derived from the plasma mrmbrane, the amounts of lipidpresant in the DRMS varicd contaterably between detergents.

Hi, thanks a lot for asking this technical experimental question. As You have asked to paraphrase…

Answered: Please paraphrase how both experiments…

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Biochemistry

Please paraphrase how both experiments (from fig.2 and 3) are asking differing questions

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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