Purification and characterization of hen oviduct microsomal signal peptidase.
Hen oviduct signal peptidase requires only two proteins for proteolysis of fully synthesized secretory precursor proteins in vitro: one with a molecular mass of 19 kilodaltons (kDa) and one which is a glycoprotein whose mass varies from 22 to 24 kDa depending on the extent of glycosylation. Purified signal peptidase has been analyzed both as part of an active catalytic unit and after electroelution of the individual proteins out of a preparative polyacrylamide gel. The multiple forms of the glycoprotein component of signal peptidase bind to concanavalin A and are shown to be derived from the same polypeptide backbone. Removal of their oligosaccharides by digestion with N-glycanase converts these proteins to a single 19.5-kDa polypeptide. The glycoproteins all exhibit very similar profiles following individual digestion with trypsin and separation of the resulting peptides by reverse-phase high-performance liquid chromatography. In addition, sequence analysis of selected peptides from corresponding regions in chromatograms representing each form of the glycoprotein reveals the same amino acid sequences. The 19-kDa signal peptidase protein does not bind concanavalin A, has a distinct tryptic peptide map from that of the glycoprotein, and appears to share no amino acid sequences in common with the glycoprotein. Its copurification on a concanavalin A-Sepharose column indicates that it must interact directly with the glycoprotein subunit.
Baker
R K
RK
Department of Biochemistry, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103.
Lively
M O
MO
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002845:Chromatography; D002846:Chromatography, Affinity; D017886:Durapatite; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification; D006882:Hydroxyapatites; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010057:Oviducts / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D012697:Serine Endopeptidases; D014357:Trypsin
D006023:Glycoproteins; D006882:Hydroxyapatites; D008565:Membrane Proteins; D010446:Peptide Fragments; D017886:Durapatite; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; C026515:type I signal peptidase