Structural studies on a 75-kDa glycoprotein isolated from porcine gastric mucosal membranes: close homology with the 78-kDa glucose-regulated family of proteins.
A 75-kDa glycoprotein (P75) has been purified to homogeneity from washed membranes isolated from the corpus of porcine gastric mucosa. The purification procedure employed chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative polyacrylamide gel electrophoresis. Reversed-phase microbore high-performance liquid chromatography was employed to fractionate and purify a number of tryptic peptides generated from approximately 1100 pmol purified P75. The use of reversed-phase microbore (2.1 mm internal diameter) columns facilitated the purification of subnanomole amounts of polypeptides in small volumes (40-60 microliter) suitable for loading onto the gas-phase sequencer without further concentration. N-Terminal amino acid sequence analyses were performed on the intact polypeptide and on 13 tryptic peptides and one Staphylococcus protease V8 peptide, yielding 170 unique assignments; this corresponds to approximately 26% of the molecule. A comparison of this amino acid sequence information with the cDNA-deduced primary structure of a 70-kDa heat-shock-related protein (P72), which is expressed in normal rat liver reveals that these protein sequences are almost identical, differing in only 1 of the 170 positions positively assigned thus far. The probable correspondence of P72 with the 78-kDa glucose-regulated protein (GRP78) isolated from hamster fibroblasts has been reported.
Baldwin
G S
GS
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Parkville, Victoria, Australia.
Chandler
R
R
Seet
K L
KL
Weinstock
J
J
Grego
B
B
Rubira
M
M
Mortiz
R L
RL
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D005753:Gastric Mucosa / Q000032:analysis*; D006023:Glycoproteins / Q000302:isolation & purification; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D013552:Swine
D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins