Porcine cerebroside sulfate activator: further structural characterization and disulfide identification.
Cerebroside sulfate activator (CS-Act) is a small compact protein which binds and solubilizes certain glycosphingolipids. Following the recent publication of the purification and preliminary sequence of pig kidney CS-Act [Fluharty, A.L., Katona, Z., Meek, W.E., Frei, K., & Fowler, A.V. (1992) Biochem. Med. Metab. Biol. 47, 66-85], we now report the primary sequence of the C-terminal portion of this protein and the assignment of the three disulfide bonds. Cyanogen bromide (CNBr) treatment of native CS-Act produced three major and several minor peptide fragments. Analysis of one HPLC-purified fragment revealed the C-terminus 14 amino acid sequence. This established the length of the native protein at 79 residues. In conjunction with the sequence data for one other major HPLC-purified CNBr fragment, it could be concluded that the three intrachain disulfide bonds were located at half-cystine residues 4 and 77, 7 and 71, and 36 and 47. Mass spectrometry (fast atom bombardment and electrospray ionization) showed the molecular weight of the major component of the CS-Act preparation to be 9720.5 Da, which was in close agreement with the calculated mass of the 79 amino acid peptide with five covalently attached sugar residues and three internal disulfide bonds. The mass spectrometric molecular weight measurements also showed that the CS-Act preparation possessed microheterogeneity in its carbohydrate moiety, as less intense signals corresponded to species containing (in decreasing order of abundance) two, one, four, and three sugar residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Stevens
R L
RL
Department of Biological Chemistry, UCLA School of Medicine 90024.
Faull
K F
KF
Conklin
K A
KA
Green
B N
BN
Fluharty
A L
AL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003488:Cyanogen Bromide; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013552:Swine
D000596:Amino Acids; D006023:Glycoproteins; D010446:Peptide Fragments; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D003488:Cyanogen Bromide