Formation and isomerization of disulfide bonds in proteins: Protein disulfide-isomerase

Abstract

Publisher Summary Protein disulfide-isomerase (PDI) catalyzes the formation of native proteins from the reduced denatured state. When incubated in the presence of a thiol compound, PDI catalyzes the regain of native ribonuclease structure from the scrambled ribonuclease, with concomitant return of activity toward RNA. This assay is based on a patently nonphysiological substrate. It is very sensitive and has permitted the study of PDI activity in a number of contexts, making it possible to propose a physiological role for this activity. The chapter describes the preparation of scrambled ribonuclease from the beef pancreatic ribonuclease A, which contains a complex mixture of various molecular weight components. The substrate, scrambled ribonuclease, is essentially inactive in the hydrolytic cleavage of high-molecular-weight RNA, having about 2% of the activity of native ribonuclease. The action of PDI in catalyzing the interchange of inter- and intramolecular disulfides in scrambled ribonuclease results in the regain of the native disulfide pairing, native conformation, and concomitant return of ribonuclease activity against RNA. Thus, the activity of protein disulfide-isomerase is assayed by a time-course incubation during which aliquots are removed and ribonuclease activity toward RNA is measured. Protein disulfide-isomerase is very widely distributed and has been detected in most vertebrate tissues, although detailed studies have been confined to the enzyme from the liver.