Preparation of the cerebroside sulfate activator (CSAct or saposin B) from human urine.
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Fluharty AL, Lombardo C, Louis A, Stevens RL, Whitelegge J, Waring AJ, To T, Fluharty CB, Faull KF
Preparation of the cerebroside sulfate activator (CSAct or saposin B) from human urine.
Mol Genet Metab. 1999 Nov;68(3):391-403.
- PubMed ID
- 10562467 [ View in PubMed]
- Abstract
The purification of cerebroside sulfate activator (CSAct) or saposin B from pooled human urine is described. Urinary proteins are concentrated by ammonium sulfate precipitation. A suspension of the precipitate is heat-treated and the heat-stable proteins are fractionated through a series of chromatographic steps. An initial concanavalin A column retains little of the CSAct activity, but is important for subsequent purification. Passing the Con A effluent directly onto an octyl Sepharose column removes the protein of interest which is recovered by affinity elution with octyl glucoside. Subsequent ion-exchange and gel filtration chromatographies yield a protein of 80-90% purity, although it is sometimes necessary to repeat one or more steps. A small amount of CSAct can sometimes be recovered from the initial Con A Sepharose column by methyl mannoside elution and purified by a parallel chromatographic protocol. Mass spectral analysis suggests that the final material is a mixture of two major and several minor glycoforms of a 79 amino acid protein with the structure predicted from the human prosaposin cDNA by truncation of both N- and C-terminal regions. Sugar analysis revealed the presence of glucosamine, mannose, and fucose, consistent with the major isoforms bearing a five-sugar Man(2)GluNac(2)Fuc or a single GluNac substituent. The human urinary material is similar to the previously characterized pig kidney protein in most respects, but varies in some details.