Limited tryptic digestion near the amino terminus of bovine liver rhodanese produces active electrophoretic variants with altered refolding.
When the enzyme rhodanese was partially digested by immobilized trypsin, it retained greater than 50% of its original activity although less than 10% of the undigested enzyme remained. The predominant daughter species were two 31-kDa polypeptides whose amino termini corresponded to either residue 44 or 45 of the enzyme's sequence. Following digestion, charged species were isolated by ion exchange chromatography. Denaturing electrophoresis revealed that a 4-kDa peptide remained associated with the 31-kDa fragment. This 4-kDa peptide appears to correspond to the amino-terminal 45 residues of rhodanese. Further proteolysis gave a 2.5-kDa peptide that dissociated under non-denaturing conditions without apparent change in migration of the 31-kDa fragment on SDS gels. Refolding of undigested, urea-denatured rhodanese restored much of its activity. Similar treatment of rhodanese following limited tryptic digestion resulted in no regain of activity. Refolding of a mixture of intact and digested rhodanese resulted in regain of activity appropriate for the amount of intact rhodanese in the sample, indicating that clipped rhodanese does not inhibit refolding of intact rhodanese. It is concluded that portions of the amino terminus of rhodanese are important in the enzyme's folding, but are not essential for the enzyme's sulfurtransferase activity.
Merrill
G A
GA
Department of Clinical Investigation, Brooke Army Medical Center, Ft. Sam Houston, Texas 78234-6200.
Butler
M
M
Horowitz
P M
PM
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D013884:Thiosulfate Sulfurtransferase; D014357:Trypsin