The cerebroside sulfate activator from pig kidney: Purification and molecular structure

Fluharty, Arvan L.; Katona, Zoltan; Meek, William E.; Frei, Karen; Fowler, Audrée V.

doi:10.1016/0885-4505(92)90009-n

Cited by 21 publications

(29 citation statements)

References 24 publications

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“…Sap B was also purified from pig kidney according to a published protocol (20). The purity of the Sap B preparations was verified by N-terminal sequence analysis, SDS-PAGE, and electrospray mass spectrometry (5).…”

Section: Sap B Preparationmentioning

confidence: 99%

Saposin B binds and transfers phospholipids

Ciaffoni

Tatti

Boe

et al. 2006

Journal of Lipid Research

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Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids.

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Section: Sap B Preparationmentioning

confidence: 99%

Saposin B binds and transfers phospholipids

Ciaffoni

Tatti

Boe

et al. 2006

Journal of Lipid Research

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“…Five rhASA batches purified from the secretions of transfected Chinese hamster ovary cells were kindly provided by Zymenex A/S (Hillerød, Denmark) (9). Native Sap B was purified from pig kidney and human urine, respectively (11). Recombinant human Saps A, B, C, and D were produced using the Pichia pastoris expression system (12,13).…”

Section: Methodsmentioning

confidence: 99%

Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy

Matzner

Breiden

Schwarzmann

et al. 2009

Journal of Biological Chemistry

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“…Native CSAct was purified from pig kidney as described previously (11). The preparation contained predominantly a glycosylated 79 amino acid residue protein and has been characterized extensively by chromatographic and mass spectrometric analysis (23), with biological activity demonstrated by in vitro and in vivo assays (21,24,25).…”

Section: Csact and Saposins A C And Dmentioning

confidence: 99%

“…A microtized version of an established procedure (11,21) 3 l, 1 g/l). The reaction was commenced by the addition of ASA enzyme solution (3 l, 250 U/ml).…”

Section: Csact-potentiated Asa Assaymentioning

confidence: 99%

“…However, assays using natural sulfatide substrate are more complicated, because of the poor water solubility of monomeric sulfatide and its propensity to form aggregates. In this case, ASA activity can be measured using detergent-solubilized sulfatide (10), whereas CSAct activity can be measured using CSAct-solubilized sulfatide (11). As done previously in the UCLA laboratory, both of these procedures involve measuring the rate of inorganic sulfate formation.…”

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confidence: 99%

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A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

Norris

Whitelegge

Yaghoubian

et al. 2005

Journal of Lipid Research

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A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the nonhydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.

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The cerebroside sulfate activator from pig kidney: Purification and molecular structure

Cited by 21 publications

References 24 publications

Saposin B binds and transfers phospholipids

Saposin B binds and transfers phospholipids

Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

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