Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase.

1. The selective release of protein disulphide-isomerase from dog pancreas and rat liver microsomal membranes was studied to throw light on the mechanisms of retention of this enzyme within the endoplasmic reticulum, and in order to prepare microsomal membranes specifically depleted of the enzyme. 2. Protein disulphide-isomerase was quantitatively released from dog pancreas microsomal membranes by washing at pH 9 and above, as demonstrated both by enzyme assay and by immunoblotting analysis. 3. Integral membrane proteins implicated in the process of translocation and segregation of secretory proteins were retained in pH 9-washed dog pancreas microsomal membranes. 4. After pH 9 washing, dog pancreas microsomal membranes were fully active in the translocation, segregation and processing of nascent secretory proteins; these membranes therefore provide a useful experimental system for testing the action of protein disulphide-isomerase on nascent secretory proteins. 5. Protein disulphide-isomerase was not released from rat liver microsomal membranes by pH 9 washing, and was much less readily released from these membranes by sonication, washing etc. than from dog pancreas microsomal membranes. 6. The mechanism of retention of protein disulphide-isomerase, and of other resident proteins of the lumen of the endoplasmic reticulum, is discussed in the light of these findings.

Role of protein disulphide-isomerase in the expression of native proteins.

Formation of native disulphide bonds is a post-translational modification associated with the folding and assembly of secretory proteins. The process is catalysed within the lumen of the endoplasmic reticulum by the enzyme protein disulphide-isomerase (PDI), which is abundant in secretory cells and catalyses thiol: protein-disulphide interchange in vitro with very broad protein substrate specificity. The presence of PDI within microsomal vesicles is essential for efficient and rapid cotranslational disulphide bond formation during protein synthesis in vitro. The sequence of PDI is now known from several species, and shows the presence of two domains closely homologous to thioredoxins. Chemical modification data confirm the role of the thioredoxin domains in thiol:disulphide interchange activity, and structural models of these domains can be built based on homology with thioredoxin. Thus PDI is both strongly implicated in the process of native protein folding in vivo and well characterized at the molecular level. In addition to its disulphide-isomerase activity, PDI participates as a component of the enzyme prolyl-4-hydroxylase, and further functions have also been proposed.

Induction of expression of protein disulphide-isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide.

Protein disulphide-isomerase (PDI) activity, and the level of immunodetectable PDI protein, were monitored in splenic lymphocytes and in BCL1 cells during culture in the presence of various activating factors. Bacterial lipopolysaccharide stimulated induction of PDI in splenic B cells and BCL1 cells. The time-course and specificity of induction indicated that the increase in expression of PDI is closely coupled to the final stages of B cell differentiation into antibody-producing plasma cells. The system will prove valuable in studies on the control of expression of PDI.