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PMID: 3511473 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Purification of microsomal signal peptidase as a complex.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 83 ·No. 3 ·1986-02-00 ·Pages 581-5

Evans EA, Gilmore R, Blobel G

Abstract

We report here the purification to near homogeneity of signal peptidase from canine pancreatic microsomes. Purification was monitored using an improved post-translational 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.

MeSH Terms
Animals Centrifugation, Density Gradient Chromatography, Gel Chromatography, Ion Exchange Dogs Endopeptidases/isolation & purification Glycoproteins/isolation & purification Intracellular Membranes/enzymology Macromolecular Substances Membrane Proteins Microsomes/enzymology Pancreas/enzymology Protein Processing, Post-Translational Serine Endopeptidases
Chemicals
Glycoproteins Macromolecular Substances Membrane Proteins Endopeptidases Serine Endopeptidases type I signal peptidase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Evans E A
Gilmore R
Blobel G
References (31)
31 references, click to expand
  1. Preparation of microsomal membranes for cotranslational protein translocation.
    Methods Enzymol. 1983;96:84-93 PMID: 6656655
  2. Quantitative assay for signal peptidase.
    Methods Enzymol. 1983;96:784-94 PMID: 6361465
  3. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.
    J Cell Biol. 1975 Dec;67(3):835-51 PMID: 811671
  4. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components.
    J Cell Biol. 1975 Dec;67(3):852-62 PMID: 811672
  5. Nascent prehormones are intermediates in the biosynthesis of authentic bovine pituitary growth hormone and prolactin.
    Proc Natl Acad Sci U S A. 1977 Jun;74(6):2432-6 PMID: 267936
  6. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598-602 PMID: 271987
  7. The sodium channel from rat brain. Purification and subunit composition.
    J Biol Chem. 1984 Feb 10;259(3):1667-75 PMID: 6319405
  8. Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene.
    Proc Natl Acad Sci U S A. 1984 Jun;81(12):3708-12 PMID: 6374664
  9. Protein translocation across the endoplasmic reticulum.
    Cell. 1984 Aug;38(1):5-8 PMID: 6088076
  10. Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.
    Proc Natl Acad Sci U S A. 1984 Nov;81(21):6579-83 PMID: 6436817
  11. Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex.
    J Biol Chem. 1984 Nov 25;259(22):14059-63 PMID: 6094538
  12. Mammalian signal peptidase: partial purification and general characterization of the signal peptidase from microsomal membranes of porcine pancreas.
    J Biochem. 1984 Oct;96(4):1125-31 PMID: 6084004
  13. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes.
    J Cell Biol. 1978 May;77(2):464-87 PMID: 649658
  14. Processing of prepromelittin by subcellular fractions from rat liver.
    Biochem Biophys Res Commun. 1978 Aug 14;83(3):901-7 PMID: 708440
  15. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
  16. Intracellular protein topogenesis.
    Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496-500 PMID: 6929499
  17. Post-translational processing of full-length presecretory proteins with canine pancreatic signal peptidase.
    Ann N Y Acad Sci. 1980;343:391-404 PMID: 6994559
  18. Phospholipid is required for the processing of presecretory proteins by detergent-solubilized canine pancreatic signal peptidase.
    J Biol Chem. 1981 Mar 10;256(5):2545-50 PMID: 7007387
  19. Biosynthesis of a lysosomal enzyme. Partial structure of two transient and functionally distinct NH2-terminal sequences in cathepsin D.
    J Biol Chem. 1981 Nov 10;256(21):11224-31 PMID: 6116713
  20. Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein.
    J Cell Biol. 1981 Nov;91(2 Pt 1):545-50 PMID: 7309795
  21. Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide.
    Proc Natl Acad Sci U S A. 1982 Apr;79(7):2260-3 PMID: 7048315
  22. A bacterial secretory protein requires signal recognition particle for translocation across mammalian endoplasmic reticulum.
    J Biol Chem. 1982 Oct 25;257(20):11860-3 PMID: 6749848
  23. Protein translocation across the endoplasmic reticulum. II. Isolation and characterization of the signal recognition particle receptor.
    J Cell Biol. 1982 Nov;95(2 Pt 1):470-7 PMID: 6292236
  24. Lactose-proton symport by purified lac carrier protein.
    Biochemistry. 1982 Oct 26;21(22):5634-8 PMID: 6293552
  25. Phospholipids stabilize the interaction between the alpha and beta subunits of the solubilized receptor for immunoglobulin E.
    Biochemistry. 1982 Dec 21;21(26):6922-7 PMID: 6218820
  26. Signal peptide digestion in Escherichia coli. Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein.
    Eur J Biochem. 1982 Dec;129(1):233-9 PMID: 6761118
  27. M13 procoat and a pre-immunoglobulin share processing specificity but use different membrane receptor mechanisms.
    Proc Natl Acad Sci U S A. 1983 May;80(10):2809-13 PMID: 6344069
  28. Hen oviduct signal peptidase is an integral membrane protein.
    J Biol Chem. 1983 Aug 10;258(15):9488-95 PMID: 6348046
  29. Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope.
    J Biol Chem. 1983 Oct 10;258(19):12073-80 PMID: 6311837
  30. Cell-free translation of messenger RNA in a wheat germ system.
    Methods Enzymol. 1983;96:38-50 PMID: 6656637
  31. A rapid, sensitive, and specific method for the determination of protein in dilute solution.
    Anal Biochem. 1973 Dec;56(2):502-14 PMID: 4128882
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.83.3.581
Published
1986-02-00
Pages
581-5
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC322907
Subset
IM
Grants
NIGMS NIH HHS · GM 07982 · United States
NIGMS NIH HHS · GM 27155 · United States
NIGMS NIH HHS · GM 33419 · United States
External Links
PubMed source DOI: 10.1073/pnas.83.3.581 PMC Full Text View journal details
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