Preparation of the cerebroside sulfate activator (CSAct or saposin B) from human urine.
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.
Fluharty
A L
AL
Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California, 90024-1759, USA.
Lombardo
C
C
Louis
A
A
Stevens
R L
RL
Whitelegge
J
J
Waring
A J
AJ
To
T
T
Fluharty
C B
CB
Faull
K F
KF
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000302:isolation & purification / Q000652:urine*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000652:urine*; D006801:Humans; D006868:Hydrolysis; D007668:Kidney; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000652:urine; D011498:Protein Precursors / Q000235:genetics; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013552:Swine
D000596:Amino Acids; D002241:Carbohydrates; D020536:Enzyme Activators; D006023:Glycoproteins; D009844:Oligosaccharides; C483345:PSAP protein, human; D020033:Protein Isoforms; D011498:Protein Precursors; D049231:Saposins; D049128:Sphingolipid Activator Proteins