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.
Paver
J L
JL
Biological Laboratory, University of Kent, Canterbury, U.K.
Hawkins
H C
HC
Freedman
R B
RB
D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D010179:Pancreas / Q000201:enzymology*; D019704:Protein Disulfide-Isomerases
D008565:Membrane Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases