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

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

Basic Clinical Laboratory Techniques 6E
Basic Clinical Laboratory Techniques 6E
6th Edition
ISBN:9781133893943
Author:ESTRIDGE
Publisher:ESTRIDGE
Chapter6: Basic Clinical Chemistry
Section6.1: Introduction To Clinical Chemistry
Problem 9RQ
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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 been found to be partly insoluble (15), consistent with our results. In contrast, TfR, which in our hands largely resisted extraction with Lubrol WX, was reported to be Lubrol-soluble in MDCK cells (15). Therefore, analysis of the protein contents of DRMS from MDCK cells showed that the detergents differed considerably in their ability to enrich strongly lipid-associated proteins; i.e., they differed in thcir "DRM selectivities." Lipid Analysis of DRMS Prepared with Different Detergents. To analyze the lipid content of DRMS, MDCK and Jurkat cells were used to label either all lipids with 1"C or phospholipids with 32P. Total cell membranes (TCMS) and DRMS were obtaincd by flotation on sucrose step gradients. Lipids were extracted and resolved by TLC (Fig. 3). As observed for the protein content of DRMS derived from the plasma membrane, the amounts of lipid present in the DRMS varied considerably between detergents. To show qualitative changes in the lipid composition of DRMS compared with TCMS, lipids were grouped into sphingolipids, cholesterol, and glycerophospholipids. Sphingolipid/glycero- Compared with TCMS, DRMS from MDCK cells prepared with Triton and CHAPS showed clear enrichment of sphingo- lipids and cholesterol over glycerophospholipids. The other detergents enriched these lipids less strongly. Nevertheless, their capacity for enrichment correlated with the DRM specificity with regard to proteins, such that Brij 98 and Brij 96 caused a more pronounced enrichment of sphingolipids and cholesterol than Tween 20, Brij 58, and Lubrol WX. The same trend was observed for Jurkat cells, but notable differences exist. Contrary to MDCK cells, Brij 98 and Brij 58 enriched sphingolipids and cholesterol to a similar extent, whereas Brij 96 did not. The behaviors of Brij 58, Brij 96, and Brij 98 in MDCK and Jurkat cells illustrate that the effect of a given detergent may vary between cell types. Morcover, the enrichment of sphingolipids and cholesterol was overall less pronounced in Jurkat cells than in MDCK cells, showing that the lipid composition of DRMS prepared with a particular detergent can be cell type-dependent. Next, we analyzed the fatty acid substitution pattern of glycerophospholipids 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 mass spectrometry. Seventeen PC species, differing in the total num- cholesterol cholesterol Gluc/Galc Gluc PE PE LacC PC/PVPS SM PC/PIPS SM Gb Fig. 3. Lipid contents of DRMS from MDCK and Jurkat cells. Lipids from TCMS and DRMS from 14C- or 12p.labeled MDCK and Jurkat cells were analyzed by TLC (Upper, 1"C-labeled lipids; 12p-labeled lipids). Gluc/GalC/LacC, glucosyl/galactosyl/ PE PE- PI PIㅡ PS Lower, lactosyl ceramide; SM, sphingomyelin; Gbs pentosylglucoside; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. Asterisks denote lipids not identified by comigration with lipid standards. PS PC PC SM< SM MDCK cells Jurkat cells PNAS I May 13, 2003 | vol. 100 | no. 10 | 5797 Schuck et al. Brij 96 Brij 98 Lubrol Brij 58 CHAPS riton Brij 96 Brij 98 Lubrol Brij 58 Tween Tween TCM
Transcribed Image Text:The results with Triton agreed with earlier observations (6, 11, 20-22). Regarding Lubrol WX, calnexin had previously been found to be partly insoluble (15), consistent with our results. In contrast, TfR, which in our hands largely resisted extraction with Lubrol WX, was reported to be Lubrol-soluble in MDCK cells (15). Therefore, analysis of the protein contents of DRMS from MDCK cells showed that the detergents differed considerably in their ability to enrich strongly lipid-associated proteins; i.e., they differed in thcir "DRM selectivities." Lipid Analysis of DRMS Prepared with Different Detergents. To analyze the lipid content of DRMS, MDCK and Jurkat cells were used to label either all lipids with 1"C or phospholipids with 32P. Total cell membranes (TCMS) and DRMS were obtaincd by flotation on sucrose step gradients. Lipids were extracted and resolved by TLC (Fig. 3). As observed for the protein content of DRMS derived from the plasma membrane, the amounts of lipid present in the DRMS varied considerably between detergents. To show qualitative changes in the lipid composition of DRMS compared with TCMS, lipids were grouped into sphingolipids, cholesterol, and glycerophospholipids. Sphingolipid/glycero- Compared with TCMS, DRMS from MDCK cells prepared with Triton and CHAPS showed clear enrichment of sphingo- lipids and cholesterol over glycerophospholipids. The other detergents enriched these lipids less strongly. Nevertheless, their capacity for enrichment correlated with the DRM specificity with regard to proteins, such that Brij 98 and Brij 96 caused a more pronounced enrichment of sphingolipids and cholesterol than Tween 20, Brij 58, and Lubrol WX. The same trend was observed for Jurkat cells, but notable differences exist. Contrary to MDCK cells, Brij 98 and Brij 58 enriched sphingolipids and cholesterol to a similar extent, whereas Brij 96 did not. The behaviors of Brij 58, Brij 96, and Brij 98 in MDCK and Jurkat cells illustrate that the effect of a given detergent may vary between cell types. Morcover, the enrichment of sphingolipids and cholesterol was overall less pronounced in Jurkat cells than in MDCK cells, showing that the lipid composition of DRMS prepared with a particular detergent can be cell type-dependent. Next, we analyzed the fatty acid substitution pattern of glycerophospholipids 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 mass spectrometry. Seventeen PC species, differing in the total num- cholesterol cholesterol Gluc/Galc Gluc PE PE LacC PC/PVPS SM PC/PIPS SM Gb Fig. 3. Lipid contents of DRMS from MDCK and Jurkat cells. Lipids from TCMS and DRMS from 14C- or 12p.labeled MDCK and Jurkat cells were analyzed by TLC (Upper, 1"C-labeled lipids; 12p-labeled lipids). Gluc/GalC/LacC, glucosyl/galactosyl/ PE PE- PI PIㅡ PS Lower, lactosyl ceramide; SM, sphingomyelin; Gbs pentosylglucoside; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. Asterisks denote lipids not identified by comigration with lipid standards. PS PC PC SM< SM MDCK cells Jurkat cells PNAS I May 13, 2003 | vol. 100 | no. 10 | 5797 Schuck et al. Brij 96 Brij 98 Lubrol Brij 58 CHAPS riton Brij 96 Brij 98 Lubrol Brij 58 Tween Tween TCM
top bottom top bottom top bottom VIP17/MAL PLAP caveolin-1 stomatin Yes TIR gp114 calnexin rab-5 Lubrol WX Brij 96 Triton Fig. 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 gradients are 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 of these marker proteins confirms the graded selectivity of the detergents obscrved by analysis of the plasma membrane DRM protein content. The results with Triton agreed with earlier observations (6, 11, 20-22). Regarding Lubrol WX, calnexin had previously been found to be partly insoluble (15), consistent with our results. In contrast, TfR, which in our hands largely resisted extraction with Lubrol WX, was reported to be Lubrol-soluble in MDCK cells (15). Therefore, analysis of the protein contents of DRMS from MDCK cells showed that the detergents differed considerably in their ability to enrich strongly lipid-associated proteins; i.e., they differed in their "DRM selectivities." phospholipid and cholesterol/glycerophospholipid ratios were calculated. The fold changes of these ratios relative to the TCMS are depicted in Fig. 4. No significant changes occurred when higher detergent concentrations were used or when DRMS where prepared by pelleting from isolated membranes instead of flotation from cell homogenate (not shown). Compared with TCMS, DRMS from MDCK cells prepared with Triton and CHAPS showed clear enrichment of sphingo- lipids and cholesterol over glycerophospholipids. The other detergents enriched these lipids less strongly. Nevertheless, their capacity for enrichment correlated with the DRM specificity with regard to proteins, such that Brij 98 and Brij 96 caused a more pronounced enrichment of sphingolipids and cholesterol than Tween 20, Brij 58, and Lubrol WX. The same trend was observed for Jurkat cells, but notable differences exist. Contrary to MDCK cells, Brij 98 and Brij 58 enriched sphingolipids and cholesterol to a similar extent, whereas Brij 96 did not. The behaviors of Brij 58, Brij 96, and Brij 98 in MDCK and Jurkat cells illustrate that the effect of a given detergent may vary between cell types. Morcover, the enrichment of sphingolipids and cholesterol was overall less pronounced in Jurkat cells than in MDCK cells, showing that the lipid composition of DRMS prepared with a particular detergent can be cell type-dependent. Next, we analyzed the fatty acid substitution pattern of slyserophospholipids in different DRMS, Lipids from the float- Lipid Analysis of DRMS Prepared with Different Detergents. To analyze the lipid content of DRMS, MDCK and Jurkat cells were used to label cither all lipids with 14C or phospholipids with 32P. Total cell membranes (TCMS) and DRMS were obtained by flotation on sucrose step gradients. Lipids were extracted and resolved by TLC (Fig. 3). As observed for the protein content of DRMS derived from the plasma mrmbrane, the amounts of lipid presant in the DRMS varicd contaterably between detergents.
Transcribed Image Text:top bottom top bottom top bottom VIP17/MAL PLAP caveolin-1 stomatin Yes TIR gp114 calnexin rab-5 Lubrol WX Brij 96 Triton Fig. 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 gradients are 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 of these marker proteins confirms the graded selectivity of the detergents obscrved by analysis of the plasma membrane DRM protein content. The results with Triton agreed with earlier observations (6, 11, 20-22). Regarding Lubrol WX, calnexin had previously been found to be partly insoluble (15), consistent with our results. In contrast, TfR, which in our hands largely resisted extraction with Lubrol WX, was reported to be Lubrol-soluble in MDCK cells (15). Therefore, analysis of the protein contents of DRMS from MDCK cells showed that the detergents differed considerably in their ability to enrich strongly lipid-associated proteins; i.e., they differed in their "DRM selectivities." phospholipid and cholesterol/glycerophospholipid ratios were calculated. The fold changes of these ratios relative to the TCMS are depicted in Fig. 4. No significant changes occurred when higher detergent concentrations were used or when DRMS where prepared by pelleting from isolated membranes instead of flotation from cell homogenate (not shown). Compared with TCMS, DRMS from MDCK cells prepared with Triton and CHAPS showed clear enrichment of sphingo- lipids and cholesterol over glycerophospholipids. The other detergents enriched these lipids less strongly. Nevertheless, their capacity for enrichment correlated with the DRM specificity with regard to proteins, such that Brij 98 and Brij 96 caused a more pronounced enrichment of sphingolipids and cholesterol than Tween 20, Brij 58, and Lubrol WX. The same trend was observed for Jurkat cells, but notable differences exist. Contrary to MDCK cells, Brij 98 and Brij 58 enriched sphingolipids and cholesterol to a similar extent, whereas Brij 96 did not. The behaviors of Brij 58, Brij 96, and Brij 98 in MDCK and Jurkat cells illustrate that the effect of a given detergent may vary between cell types. Morcover, the enrichment of sphingolipids and cholesterol was overall less pronounced in Jurkat cells than in MDCK cells, showing that the lipid composition of DRMS prepared with a particular detergent can be cell type-dependent. Next, we analyzed the fatty acid substitution pattern of slyserophospholipids in different DRMS, Lipids from the float- Lipid Analysis of DRMS Prepared with Different Detergents. To analyze the lipid content of DRMS, MDCK and Jurkat cells were used to label cither all lipids with 14C or phospholipids with 32P. Total cell membranes (TCMS) and DRMS were obtained by flotation on sucrose step gradients. Lipids were extracted and resolved by TLC (Fig. 3). As observed for the protein content of DRMS derived from the plasma mrmbrane, the amounts of lipid presant in the DRMS varicd contaterably between detergents.
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