Multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme

Nesmeyanova, M. A.; Motlokh, O B; Kolot, Mikhail; Kulaev, I. S.

doi:10.1128/jb.146.2.453-459.1981

Cited by 15 publications

(2 citation statements)

References 22 publications

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“…It should be noted that a variety of studies have reported the presence of isozymes of Ec ALP that have different N‐terminal residues, which are generated by post‐translational processing, after removal of a signal peptide, during maturation. 52 , 53 , 54 , 55 These studies employed signal peptides for the secretion of ALPs into periplasm space where the disulfide bond formation in monomers is mediated with Dsb proteins under oxidative environments. On the other hand, in this study, the recombinant Ec ALP was expressed without signal peptide and purified with N‐terminal histidine‐tag.…”

Section: Resultsmentioning

confidence: 99%

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

et al. 2021

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Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers.However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered singlemolecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k cat , and the rate constant of productive binding, k on , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.

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Section: Resultsmentioning

confidence: 99%

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

et al. 2021

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“…There are indications that part of E. coli and N . crassa cell polyphosphatase is also connected with the cytoplasmic membrane (Nesmeyanova et al, 1975;Kulaev et al, 1972).…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Andreeva

Okorokov

1993

Yeast

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Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM-MgCl2, 0.5 mM-EDTA and 0.1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at -20 degrees C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3-208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, Km values were 1.7 x 10(-4), 1.5 x 10(-5) and 8.8 x 10(-7) M respectively. Polyphosphatase was most active and stable at pH 6.0-8.0. The enzyme showed maximal activity at 50 degrees C. The time of half inactivation of polyphosphatase at 40, 45 and 50 degrees C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca2+ or Cu2+, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co2+> Mg2+ > Mn2+ > Fe2+ > Zn2+. Polyphosphatase was completely inhibited by 1 mM-ammonium molybdate and 50 microM-Zn2+ or Cu2+ (in the presence of Mg2+).

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Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region 1 1Edited by A. R. Fersht

Karamyshev

Karamysheva

Kajava³

et al. 1998

Journal of Molecular Biology

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Multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme

Cited by 15 publications

References 22 publications

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region 1 1Edited by A. R. Fersht

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