Purification and characterization of the low molecular weight protein tyrosine phosphatase, Stp1, from the fission yeast Schizosaccharomyces pombe

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Abstract

Genetic screening in fission yeast has identified a gene named stpl+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low Mr PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low Mr PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30°C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low Mr PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low Mr PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

Original languageEnglish (US)
Pages (from-to)10560-10568
Number of pages9
JournalBiochemistry
Volume34
Issue number33
StatePublished - 1995
Externally publishedYes

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Protein Tyrosine Phosphatases
Schizosaccharomyces
Yeast
Purification
Molecular Weight
Molecular weight
Phosphates
Phosphoric Monoester Hydrolases
Kinetics
Proteins
Substrates
cdc25 Phosphatases
Genes
Yeasts
Dual-Specificity Phosphatases
Phosphoserine
Electrospray ionization
Phosphotyrosine
Electrospray Ionization Mass Spectrometry
Protein Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Purification and characterization of the low molecular weight protein tyrosine phosphatase, Stp1, from the fission yeast Schizosaccharomyces pombe",
abstract = "Genetic screening in fission yeast has identified a gene named stpl+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42{\%} identical to members of the low molecular weight protein tyrosine phosphatases (low Mr PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low Mr PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30°C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low Mr PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low Mr PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.",
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T1 - Purification and characterization of the low molecular weight protein tyrosine phosphatase, Stp1, from the fission yeast Schizosaccharomyces pombe

AU - Zhang, Zhong-Yin

PY - 1995

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N2 - Genetic screening in fission yeast has identified a gene named stpl+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low Mr PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low Mr PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30°C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low Mr PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low Mr PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

AB - Genetic screening in fission yeast has identified a gene named stpl+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low Mr PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low Mr PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30°C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low Mr PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low Mr PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

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