The latency of rat liver microsomal protein disulphide-isomerase

Abstract

Protein disulphide-isomerase (PDI) activity was not detectable in freshly prepared rat liver microsomes (microsomal fraction), but became detectable after treatments that damage membrane integrity, e.g. sonication, detergent treatment or freezing and thawing. Maximum activity was detectable after sonication. Identical latency was observed in microsomes prepared by gel filtration and in those prepared by high-speed centrifugation. PDI activity was latent in all particulate subcellular fractions, but not latent in the high-speed supernatant. When all fractions were sonicated to expose total PDI activity, PDI was found at highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum. Washing of microsomes under various conditions that removed peripheral proteins and, in some cases, bound ribosomes did not remove significant quantities of PDI, nor did it affect the latency of PDI activity. Treatment of microsomes with proteinases, under conditions where the permeability barrier of the microsomal vesicles was maintained intact, did not inactivate PDI significantly or affect its latency. PDI was very readily solubilized from microsomal vesicles by low concentrations of detergents, which removed only a fraction of the total microsomal protein. In all these respects, PDI resembled nucleoside diphosphatase, a marker peripheral protein of the luminal surface of the endoplasmic reticulum, and differed from NADPH: cytochrome c reductase, a marker integral protein exposed at the cytoplasmic surface of the membrane. The data are compatible with a model in which PDI is loosely associated with the luminal surface of the endoplasmic reticulum, a location consistent with the proposed physiological role of the enzyme as catalyst of formation of native disulphide bonds in nascent and newly synthesized secretory proteins.

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

“…As noted above, there is already abundant evidence that PDI can act in vitro to catalyse disulphide bond formation in Ig folding and assembly, and that PDI is present at high levels in lymphoma-derived cell lines actively secreting immunoglobulins. PDI is located within the lumen of the endoplasmic reticulum [25] the site of immunoglobulin folding and assembly [26]. Furthermore, PDI can be chemically cross-linked to nascent immunoglobulins in hybridoma cells [27].…”

Formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus 40 Vp1 in the cytoplasm

“…An alternative possibility, that the disulfides are catalyzed artifactually by the ER-resident PDI upon subcellular fractionation (32) and that this activity is inhibited by NEM, is unlikely, because the Vp1 oligomers also were detected (i) when fractions were treated with NEM and directly analyzed by immunoblotting (Fig. 2 A-C), (ii) in fractions prepared with lysis buffer containing NEM, and (iii) in a soluble cytoplasmic fraction prepared by hypotonic cell lysis (data not shown) that is not expected to contain PDI activity (32,33). Therefore, Vp1 folding and oligomer assembly, accompanied by redox reactions, likely take place in the cytosol, outside the ER-Golgi system.…”

Studies on the formation of intrachain disulphide bonds in newly biosynthesised bovine prolactin

“…Protein disulphide-isomerase activity was measured as described by Lambert and Freedman [22], in which re-activation of 'scrambled' ribonuclease is monitored by dual-wavelength spectroscopic assay of ribonuclease. The incubation buffer was 0.25 M sucrose, 50 mM Tris/HCl pH 7.5, 5 mM EDTA.…”