The protein disulfide isomerase catalyzed reduction of insulin by glutathione is inhibited by peptides of various length and amino acid composition. Peptide inhibitors are competitive against insulin and noncompetitive against GSH, consistent with a sequential rather than a double displacement mechanism. Peptides of unrelated primary sequence that do not contain cysteine inhibit the GSH-insulin transhydrogenase activity of PDI, and the affinity of these peptides toward the enzyme is largely dependent on the peptide length rather than composition, hydrophobicity, or charge. Cysteine-containing peptides are 4-8-fold better inhibitors than non-cysteine-containing peptides of the same length, suggesting a cysteine-specific component to the interaction with the enzyme. Oxidized insulin chain B also inhibits the oxidative folding of reduced ribonuclease in a glutathione redox buffer with an inhibition constant that is comparable to that observed for the inhibition of insulin reduction, suggesting a similar if not identical binding site for the catalysis of oxidative protein folding and the reduction of insulin.

The reversible denaturation of protein disulfide isomerase proceeds through intermediates that are stabilized by interaction with guanidine hydrochloride. At pH 7.5, the equilibrium denaturation by urea is completely reversible and the transition can be reasonably well-described by a two-state model involving only native and denatured forms. In comparison, the equilibrium denaturation by guanidine hydrochloride occurs in two distinct steps. In the presence of a low constant amount of guanidine hydrochloride (0.5-1.4 M), urea denaturation also becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by specific interaction with guanidine hydrochloride but not by high concentrations of other salts or other denaturants.Protein disulfide isomerase (PDI; EC 5.3.4.1) is a multifunctional protein (Mr = 57,000) that is located in the lumen of the endoplasmic reticulum where it is thought to catalyze thioldisulfide exchange reactions that are essential for the posttranslational formation of disulfide bonds in newly synthesized proteins (1-6). The primary sequence of PDI shows two internally homologous domains (7) that contain the two active site regions of each monomer. One domain is located near the N terminus and the other is near the C terminus. Sigma. Dithiothreitol (DTT) was purchased from Boehringer Mannheim. Gdn-HCl was sequanal grade from Pierce. Urea (ultra pure) was from ICN. Urea solutions were prepared immediately before use. Glass-distilled deionized water was used for all experiments.PDI was prepared from fresh bovine liver by the method of Lambert and Freedman (13). The purity of the enzyme was >95% as judged by polyacrylamide gel electrophoresis. The enzyme (1.5-2 mg/ml) was stored at -20°C in 20 mM sodium phosphate (pH 6.3). HPLC on a DEAE 5WP (Waters) anion-exchange column (eluted with a linear gradient of0-0.5 M NaCl over 30 min) or gel filtration on a Bio-Sil SEC250 (Bio-Rad) column revealed two major PDI species in a 1:0.7 ratio. Both peaks had PDI activity, both proteins migrated as a single 57-kDa band during SDS/PAGE under reducing and nonreducing conditions, and the N-terminal 10 residues of both species were identical to the sequence of PDI. Two forms of PDI that are resolved by gel-filtration HPLC have been reported previously and attributed to proteolysis near the C terminus (14); however, the suggested C terminus of one ofthe two peaks could not be found in the deduced cDNA sequence of PDI. The two forms of PDI appear to represent monomeric and dimeric species in which a metastable dimer without intermolecular disulfides is induced by freezing in phosphate buffer (M. Kruzel and H.F.G., unpublished observations). Overnight incubation of the preparation at pH 7.5 and 22°C results in essentially complete (>90%) conversion of the dimer to the monomer; under the conditions of our experiments, the PDI is monomeric. In addition, Gdn HCl denaturation profiles for the two forms of PDI isolated from HPLC are identical to each other and identical to those of th...

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Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI). It is found in human serum within 4-6 h following MI. One of us has shown [Morjana (1998) Biotechnol. Appl. Biochem. 28, 105-111] that MI patient serum TnI is cleaved at the N- and C-terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT). In the present study, we have generated C-terminal truncated TnI fragments and studied their immunological and biochemical properties. Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr. The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI153). Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI88, residues 6-96). TnI153 has higher immunological activity than that of rTnI and lower activity than that of TnI88, as judged by the Stratus II TnI Immunoassay. TnI153 exhibits biochemical and immunological properties similar to those of intact TnI. It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT. TnC enhances the immunological activity of TnI153, but has little effect on the activity of TnI88. The TnI153-TnC complex exhibits higher immunological activity than rTnI-TnC and TnI88-TnC, and much higher activity than free rTnI, TnI153 and TnI88. The presence of TnT has no effect on the immunological activity of the TnI153-TnC complex, suggesting that the addition of TnT does not interfere with TnI153 recognition by TnI monoclonal antibodies. Free TnI153 and TnI88 degrade rapidly in human serum. TnC protects TnI153 from proteolytic degradation, but offers less protection for TnI88. The TnI88-TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 degrees C. However, there was no loss in the immunological activity of the TnI153-TnC complex under the same conditions. A cTnI fragment (TnI80, residues 1-80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI88.

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