Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation.
The hamster gene encoding the 78-kDa glucose-regulated protein (Grp78) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. After induction with isopropyl beta-D-thiogalactopyranoside, the recombinant Grp78 was purified to homogeneity by affinity column chromatography of the fusion protein followed by thrombin cleavage. The purified recombinant protein was compared with liver Grp78 for its ability to interact with ATP. Like liver Grp78, the recombinant protein contained a weak ATPase activity and a Ca(2+)-stimulated autophosphorylation activity. However, unlike liver Grp78, in which the autophosphorylation reaction is stimulated less than 50% by CaCl2, the reaction with the recombinant Grp78 was stimulated about 15-fold in the presence of Ca2+. Although the liver protein showed at least four isoforms after two-dimensional gel electrophoresis, the recombinant Grp78 had one major species corresponding to the most basic form seen in liver. Both the liver Grp78 and the recombinant protein existed primarily as monomers and dimers. A small amount of oligomers was also present in the liver Grp78. When either protein was incubated with ATP, there was a conversion of the higher molecular weight species to the monomeric form.
Carlino
A
A
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Toledo
H
H
Skaleris
D
D
DeLisio
R
R
Weissbach
H
H
Brot
N
N
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases