Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase
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| 024 | 7 | 0 | |a 10.1515/BC.2004.154 |2 doi |
| 035 | |a (NATIONALLICENCE)gruyter-10.1515/BC.2004.154 | ||
| 245 | 0 | 0 | |a Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase |h [Elektronische Daten] |c [Melanie Kölzer, Klaus Ferlinz, Oliver Bartelsen, Silvia Locatelli Hoops, Florian Lang, Konrad Sandhoff] |
| 520 | 3 | |a Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo. A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C. | |
| 540 | |a ©2004 by Walter de Gruyter Berlin New York | ||
| 690 | 7 | |a Biochemistry |2 nationallicence | |
| 690 | 7 | |a Molecular biology |2 nationallicence | |
| 690 | 7 | |a Cellular biology |2 nationallicence | |
| 690 | 7 | |a acid sphingomyelinase |2 nationallicence | |
| 690 | 7 | |a saposin-homologous domain |2 nationallicence | |
| 690 | 7 | |a site-directed mutagenesis |2 nationallicence | |
| 690 | 7 | |a water-lipid interface |2 nationallicence | |
| 700 | 1 | |a Kölzer |D Melanie |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | |
| 700 | 1 | |a Ferlinz |D Klaus |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | |
| 700 | 1 | |a Bartelsen |D Oliver |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | |
| 700 | 1 | |a Hoops |D Silvia Locatelli |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | |
| 700 | 1 | |a Lang |D Florian |u Institut für Physiologie, Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany |4 aut | |
| 700 | 1 | |a Sandhoff |D Konrad |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | |
| 773 | 0 | |t Biological Chemistry |d Walter de Gruyter |g 385/12(2004-12-01), 1193-1195 |x 1431-6730 |q 385:12<1193 |1 2004 |2 385 |o bchm | |
| 856 | 4 | 0 | |u https://doi.org/10.1515/BC.2004.154 |q text/html |z Onlinezugriff via DOI |
| 908 | |D 1 |a research article |2 jats | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1515/BC.2004.154 |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Kölzer |D Melanie |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Ferlinz |D Klaus |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Bartelsen |D Oliver |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Hoops |D Silvia Locatelli |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Lang |D Florian |u Institut für Physiologie, Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Sandhoff |D Konrad |u Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Biological Chemistry |d Walter de Gruyter |g 385/12(2004-12-01), 1193-1195 |x 1431-6730 |q 385:12<1193 |1 2004 |2 385 |o bchm | ||
| 900 | 7 | |b CC0 |u http://creativecommons.org/publicdomain/zero/1.0 |2 nationallicence | |
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| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-gruyter | ||