Identification of a Cysteine Residue Conferring Sensitivity of
a Maize â-Glukosidase to Silver Ion and Alkylation Treatment.

D 2001

Identification of a Cysteine Residue Conferring Sensitivity of a Maize â-Glukosidase to Silver Ion and Alkylation Treatment.

SLANÝ, Michal; Jan ZOUHAR a Břetislav BRZOBOHATÝ

Základní údaje

Originální název

Identification of a Cysteine Residue Conferring Sensitivity of a Maize â-Glukosidase to Silver Ion and Alkylation Treatment.

Autoři

SLANÝ, Michal; Jan ZOUHAR a Břetislav BRZOBOHATÝ

Vydání

Brno, V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001, s. 29-29, 2001

Nakladatel

Masarykova univerzita v Brně

Další údaje

Jazyk

angličtina

Typ výsledku

Stať ve sborníku

Obor

Genetika a molekulární biologie

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14310/01:00006022

Organizační jednotka

Přírodovědecká fakulta

ISBN

80-210-2538-7

Klíčová slova anglicky

Maize b-glucosidase; cysteine residues; thioredoxin fusion

Štítky

cysteine residues, Maize b-glucosidase, thioredoxin fusion

Změněno: 27. 5. 2003 15:09, prof. RNDr. Břetislav Brzobohatý, CSc.

Anotace

V originále

The maize Zm-p60.1 gene encodes a b-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. High sensitivity of the enzyme to inhibition by Ag+ or Hg2+ ions together with loss of enzyme activity upon treatment with alkylating agents indicates that a free sulfhydryl group is required for enzyme activity. Site directed mutagenesis followed by enzyme kinetic analysis suggested that cysteine residue in position 479 (C479) provides the sulfhydryl group required for enzyme activity (1). Here we report data confirming that C479 is the target for Ag+ and alkylation agent action. When the wild type enzyme was incubated in the presence of 0.5 to 128 mM silver nitrate, almost complete loss of activity was observed at 32 mM AgNO3. Mutations leading to substitutions of C479 by alanine (A), serine (S), arginine (R) or aspartic acid (D) residues resulted in a dramatic drop in sensitivity of the mutated enzyme to Ag+ inhibition. Further, treatment of the wild type enzyme with an alkylating agent iodoacetic acid resulted in a loss of enzyme activity while enzyme activity in C479A mutant was not affected by the alkylation treatment. Interestingly, as revealed by native PAGE followed by western blot analysis, both Ag+ and alkylation treatment led to dimer dissociation. No dimer dissociation was detected in the mutants under identical conditions. Thus, inhibition of Zm-p60.1 enzyme activity by Ag+ and/or alkylation treatment might reflect dissociation of active dimer into inactive subunits triggered by binding of a bulky group to C479.

Návaznosti

MSM 143100008, záměr

Název: Genomy a jejich funkce

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Genomy a jejich funkce

VS96096, projekt VaV

Název: Laboratoř molekulární fyziologie rostlin

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Laboratoř molekulární fyziologie rostlin

Citovat

SLANÝ, Michal; Jan ZOUHAR a Břetislav BRZOBOHATÝ. Identification of a Cysteine Residue Conferring Sensitivity of a Maize â-Glukosidase to Silver Ion and Alkylation Treatment. In V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001. Brno: Masarykova univerzita v Brně, 2001, s. 29. ISBN 80-210-2538-7.

@inproceedings{375033,
   author = {Slaný, Michal and Zouhar, Jan and Brzobohatý, Břetislav},
   address = {Brno},
   booktitle = {V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001},
   keywords = {Maize b-glucosidase; cysteine residues; thioredoxin fusion},
   language = {eng},
   location = {Brno},
   isbn = {80-210-2538-7},
   pages = {29-29},
   publisher = {Masarykova univerzita v Brně},
   title = {Identification of a Cysteine Residue Conferring Sensitivity of a Maize â-Glukosidase to Silver Ion and Alkylation Treatment.},
   year = {2001}
}

TY  - CONF
ID  - 375033
AU  - Slaný, Michal - Zouhar, Jan - Brzobohatý, Břetislav
PY  - 2001
TI  - Identification of a Cysteine Residue Conferring Sensitivity of a Maize â-Glukosidase to Silver Ion and Alkylation Treatment.
PB  - Masarykova univerzita v Brně
CY  - Brno
SN  - 8021025387
KW  - Maize b-glucosidase
KW  - cysteine residues
KW  - thioredoxin fusion
N2  - The maize Zm-p60.1 gene encodes a b-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. High sensitivity of the enzyme to inhibition by Ag+ or Hg2+ ions together with loss of enzyme activity upon treatment with alkylating agents indicates that a free sulfhydryl group is required for enzyme activity. Site directed mutagenesis followed by enzyme kinetic analysis suggested that cysteine residue in position 479 (C479) provides the sulfhydryl group required for enzyme activity (1). Here we report data confirming that C479 is the target for Ag+ and alkylation agent action. When the wild type enzyme was incubated in the presence of 0.5 to 128 mM silver nitrate, almost complete loss of activity was observed at 32 mM AgNO3. Mutations leading to substitutions of C479 by alanine (A), serine (S), arginine (R) or aspartic acid (D) residues resulted in a dramatic drop in sensitivity of the mutated enzyme to Ag+ inhibition. Further, treatment of the wild type enzyme with an alkylating agent iodoacetic acid resulted in a loss of enzyme activity while enzyme activity in C479A mutant was not affected by the alkylation treatment. Interestingly, as revealed by native PAGE followed by western blot analysis, both Ag+ and alkylation treatment led to dimer dissociation. No dimer dissociation was detected in the mutants under identical conditions. Thus, inhibition of Zm-p60.1 enzyme activity by Ag+ and/or alkylation treatment might reflect dissociation of active dimer into inactive subunits triggered by binding of a bulky group to C479.
ER  -

SLANÝ, Michal; Jan ZOUHAR a Břetislav BRZOBOHATÝ. Identification of a Cysteine Residue Conferring Sensitivity of a Maize \^a-Glukosidase to Silver Ion and Alkylation Treatment. In \textit{V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001}. Brno: Masarykova univerzita v Brně, 2001, s.~29. ISBN~80-210-2538-7.

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