Isolation and characterization of prosaposin from human milk.
Prosaposin is the precursor protein for saposins, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Prosaposin, in addition to generating the saposins in the lysosomes, also exists as an unprocessed approximately 70-kDa protein in many tissues and secretory fluids. In this study, we isolated prosaposin from human milk. Milk was fractioned by ammonium sulfate precipitation, then chromatographed with DEAE-Sephacel and G-3000 SW gel permeation-HPLC. A fraction containing prosaposin was finally purified with the anti-saposin C IgG attached affinity column. The protein staining of the purified preparation on SDS-PAGE and the Western blotting showed a single band. The sequence of the initial 10 amino acids from N-terminus of the purified protein was identical to the sequence of prosaposin deduced from cDNA. Although prosaposin itself showed beta-glucosidase activator activity at a slight degree, the activity increased much after trypsin treatment. Western blotting of the trypsin-treated sample confirmed the formation of small saposin-like bands from prosaposin by the action of trypsin.
Kondoh
K
K
Department of Neuropsychiatry, Ehime University School of Medicine, Japan.
Hineno
T
T
Sano
A
A
Kakimoto
Y
Y
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011247:Pregnancy; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D049231:Saposins; D014357:Trypsin; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors
D006023:Glycoproteins; C483345:PSAP protein, human; D011498:Protein Precursors; D049231:Saposins; D001617:beta-Glucosidase; D014357:Trypsin