Purification of Microsomal Signal Peptidase as a Complex

We report here the purification to near homogeneity of signal peptidase from canine pancreatic microsomes. Purification was monitored using an improved posttranslational assay. A 42-fold enrichment over starting membranes was achieved by selective solubilization in nonionic detergent/high-salt buffe...

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Bibliographic Details
Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 83; no. 3; pp. 581 - 585
Main Authors:	Evans, Emily Ann, Gilmore, Reid, Blobel, Günter
Format:	Journal Article
Language:	English
Published:	Washington, DC National Academy of Sciences of the United States of America 01-02-1986 National Acad Sciences
Subjects:	Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Canines Centrifugation, Density Gradient Chromatography Chromatography, Gel Chromatography, Ion Exchange Dogs Endopeptidases - isolation & purification Enzymes Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Gels Glycoproteins Glycoproteins - isolation & purification Hydrolases Intracellular Membranes - enzymology Macromolecular Substances Membrane Proteins Microsomes - enzymology Pancreas - enzymology Phosphates Protein Processing, Post-Translational Salts Serine Endopeptidases Sodium Solubilization Fissipedia Intracellular transport Carnivora Peptidase Purification Enzyme Secretion Microsome Complexes Vertebrata Mammalia Animal Property structure relationship Pancreas Dog Endoplasmic reticulum
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Summary:	We report here the purification to near homogeneity of signal peptidase from canine pancreatic microsomes. Purification was monitored using an improved posttranslational assay. A 42-fold enrichment over starting membranes was achieved by selective solubilization in nonionic detergent/high-salt buffer followed by gradient sievorptive anion and cation exchange chromatography, hydroxylapatite chromatography, gel filtration, and sucrose gradient velocity sedimentation. When examined by NaDodSO4/PAGE, the purified enzyme consisted of a complex of six polypeptides with apparent molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa subunits were shown to be glycoproteins based on their sensitivity to endoglycosidase H and their ability to bind concanavalin A. We suggest that only one subunit of this complex carries out signal peptide cleavage. The structural association of the other subunits in stoichiometric amounts may reflect their requirement in chain translocation across the microsomal membrane.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.83.3.581