Protein disulfide isomerase (PDI E.C. 5.3.4.1) catalyses the formation of disulfide bonds in secretory or cell-surface proteins during their biosynthesis. PDI activity is associated with the microsomal fraction of the cell and has been found in several mammalian tissues and in developing wheat endosperm.

The embryo and aleurone layer of germinating seeds of the wheat cultivar Galahad showed PDI activity associated with a microsome-enriched fraction of cell homogenates PDI activity in microsome-enriched fractions from aleurone layers of normally germinating seeds was significantly higher than in their abnormally germinating counterparts and diminished as germination proceeded.

Dry embryos and germinating embryos showed less PDI activity than aleurone layers. There was no significant difference in PDI activity between microsome-enriched fractions from embryos of normally and abnormally germinating seeds at 3 or 6 days of germination.

Wheat aleurone PDI was partially purified using gel filtration and had a molecular mass of 110–130 kDa and a monomeric molecular mass of ca. 57 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.

Murine monoclonal antibodies (mAbs) against Neospora caninum tachyzoites were produced to identify the cross-reactive antigens between N. caninum and Toxoplasma gondii. Ten mAbs recognizing cross-reactive antigens of both parasites were obtained and tentatively classified into 6 different groups based on their reactivity patterns in an indirect fluorescent antibody test and Western blot analysis. Three mAbs in group 1 recognized antigens located on the surface of parasites with molecular masses ranging from 28 to 76 kDa; one mAb in group 2 recognized antigens located on interior organelles of parasites with a molecular mass of 50 kDa; one mAb in group 3 recognized antigens located on interior organelles of parasites with molecular masses of 35 kDa and 14 kDa; three mAbs in group 4 recognized antigens located on interior organelles with a molecular mass of 64 kDa; one mAb in group 5 recognized antigens located on the surface of parasites with an unknown molecular mass; one mAb in group 6 recognized antigens located on the apical end of parasites with an unknown molecular mass. The mAbs in groups 1, 2, 3, and 5 showed inhibitory effects on the growth of the two parasites in vitro in a dose-dependent manner. A cDNA expression library prepared from N. caninum tachyzoite mRNA was immunoscreened with the mAb panel. Three kinds of proteins, protein disulfide isomerase (PDI), heat-shock protein 70 (HSP70), and ribosomal protein 1 (RP1), were identified as cross-reactive antigens recognized by mAbs in groups 2, 3, and 4, respectively. Some of the proteins could be useful in developing vaccines or drugs for controlling the diseases caused by the two parasites.

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the γ-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.