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PMID: 7601842 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: a caution for gene fusion studies.

Journal of bacteriology ·Vol. 177 ·No. 13 ·1995-07-00 ·Pages 3764-70

Derman AI, Beckwith J

Abstract

Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells. The alkaline phosphatase activity of E. coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended. At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice. Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm. The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm. These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing. It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely. This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins.

MeSH Terms
Alkaline Phosphatase/genetics,metabolism Amino Acid Sequence Artifacts Cell Compartmentation Cloning, Molecular Cytoplasm/enzymology,metabolism Enzyme Activation Escherichia coli/enzymology,genetics,growth & development Molecular Sequence Data Protein Conformation Protein Folding Recombinant Fusion Proteins/metabolism
Chemicals
Recombinant Fusion Proteins Alkaline Phosphatase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Derman A I
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Beckwith J
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.177.13.3764-3770.1995
Published
1995-07-00
Pages
3764-70
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC177094
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.177.13.3764-3770.1995 PMC Full Text View journal details
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