Domain structure of the 90-kDa stress protein: heparin- and antibody-binding domain.
1. To understand the physiological roles of the 90-kDa stress protein (HSP90), we investigated the heparin- and antibody-binding domains of the protein. 2. For heparin-binding sites, HSP90 was digested completely with trypsin, and the digests were applied to a heparin-Sepharose column and eluted with 1.0 M NaCl, followed by 8.0 M urea. 3. Each elutant was purified by a reverse-phase C18 column. 4. Two peptides from the NaCl-eluted fraction and no peptide from the urea-eluted fraction were purified. 5. The purified peptides were sequenced by an automated peptide sequencer. 6. One of the heparin-binding sites was present between Leu-362 and Arg-365; another was present between Leu-645 and Lys-648. 7. These two peptides were basic and considerably hydrophilic. 8. For antibody-binding sites, HSP90 was mildly digested with trypsin, electrophoresed on SDS-polyacrylamide gels and transferred to PVDF membranes. 9. The four bound of the trypsin fragments could be sequenced with a peptide sequencer. 10. There was only one antibody-binding peptide, 38 kDa, starting from Pro-2. The others showed no cross-reactivity with the antibody and started from Leu-283. 11. Therefore, the epitopes of HSP90 are present between Pro-2 and Leu-282. 12. The heparin-binding sites are present from the middle region of the HSP90 molecule, and the antigen sites are at the N-terminal domain.
Itoh
H
H
Department of Biochemistry, Akita University School of Medicine, Japan.
Tashima
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D000939:Epitopes; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D006493:Heparin; D014357:Trypsin