Preparation of the cerebroside sulfate activator (CSAct or saposin B) fromhuman urine

The purification of cerebroside sulfate activator (CSAct) or saposin B from pooled human urine is described. Urinary proteins are concentrated by ammo nium sulfate precipitation. A suspension of the precipitate is heat-treated and the heat-stable proteins are fractionated through a series of chromato graphic 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 int erest which is recovered by affinity elution with octyl glucoside, Subseque nt ion-exchange and gel filtration chromatographies yield a protein of 80-9 0% 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 S epharose column by methyl mannoside elution and purified by a parallel chro matographic protocol. Mass spectral analysis suggests that the final materi al is a mixture of two major and several minor; glycoforms of a 79 amino ac id protein with the structure predicted from the human prosaposin cDNA by t runcation of both N- and C-terminal regions. Sugar analysis revealed the pr esence of glucosamine, mannose, and fucose, consistent with the major isofo rms 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. (C) 1999 Acade mic Press.