Membrane Features and Activity of GPI-Anchored Enzymes: Alkaline Phosphatase Reconstituted in Model Membranes
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- 作者：Silvia Sesana ; Francesca Re ; Alessandra Bulbarelli ; Domenico Salerno ; Emanuela Cazzaniga ; Massimo Masserini
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：May 13, 2008
- 年：2008
- 卷：47
- 期：19
- 页码：5433 - 5440
- 全文大小：736K
- 年卷期：v.47,no.19(May 13, 2008)
- ISSN：1520-4995
文摘
The influence of membrane lipid environment on the activity of GPI-anchored enzymes was investigated with human placental alkaline phosphatase reconstituted by a detergent-dialysis technique in liposomes composed of palmitoyloleoylphosphatidylcholine, alone or in mixture with lipids enriched along with the protein within lipid rafts: cholesterol, sphingomyelin, and GM1 ganglioside. The highest Vmax was recorded for a phosphatidylcholine/10% GM1 mixture (143 
5 nmol of substrate hydrolyzed per minute per microgram of protein), while the lowest for a phosphatidylcholine/30% cholesterol mixture and for raft-mimicking 1:1:1 phosphatidylcholine/sphingolipid/cholesterol liposomes (M:M:M) (57 3 and 52 3, respectively). No significant differences in Km were detected. The protein segregation, assessed using the chemical cross-linker bis(sulfosuccinimidyl)suberate, increased with the protein:lipid ratio, within the 1:1200−1:4800 protein:lipid molar ratio range, but did not affect enzyme activity. The activity decreased when the order of the lipid bilayers was increased, higher for those containing cholesterol, as judged by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Finally, the GPI−enzyme activity was affected by membrane curvature. This result was suggested by a strong inverse correlation (Pearson’s correlation coefficient = 0.91; p < 0.0001) between activity and liposome diameter, measured by laser light scattering and ranging between 59 6 nm for a phosphatidylcholine/10% GM1 mixture (displaying the highest activity) and 188 25 nm for a phosphatidylcholine/30% cholesterol mixture and 185 23 nm for raft-mimicking liposomes (displaying the lowest activities). The activity−membrane curvature relationship was further confirmed by comparing the activity of proteoliposomes having different sizes but identical lipid compositions. These data open the possibility that the activity of GPI-anchored enzymes may be modulated by membrane microenvironment features, in particular by membrane curvature and cholesterol-enriched ordered microenvironments, such as those of lipid rafts.
5 nmol of substrate hydrolyzed per minute per microgram of protein), while the lowest for a phosphatidylcholine/30% cholesterol mixture and for raft-mimicking 1:1:1 phosphatidylcholine/sphingolipid/cholesterol liposomes (M:M:M) (57 3 and 52 3, respectively). No significant differences in Km were detected. The protein segregation, assessed using the chemical cross-linker bis(sulfosuccinimidyl)suberate, increased with the protein:lipid ratio, within the 1:1200−1:4800 protein:lipid molar ratio range, but did not affect enzyme activity. The activity decreased when the order of the lipid bilayers was increased, higher for those containing cholesterol, as judged by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Finally, the GPI−enzyme activity was affected by membrane curvature. This result was suggested by a strong inverse correlation (Pearson’s correlation coefficient = 0.91; p < 0.0001) between activity and liposome diameter, measured by laser light scattering and ranging between 59 6 nm for a phosphatidylcholine/10% GM1 mixture (displaying the highest activity) and 188 25 nm for a phosphatidylcholine/30% cholesterol mixture and 185 23 nm for raft-mimicking liposomes (displaying the lowest activities). The activity−membrane curvature relationship was further confirmed by comparing the activity of proteoliposomes having different sizes but identical lipid compositions. These data open the possibility that the activity of GPI-anchored enzymes may be modulated by membrane microenvironment features, in particular by membrane curvature and cholesterol-enriched ordered microenvironments, such as those of lipid rafts.