BRENDA - Enzyme Database show
show all sequences of 3.6.1.28

Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties

Lakaye, B.; Makarchikov, A.F.; Wins, P.; Margineanu, I.; Roland, S.; Lins, L.; Aichour, R.; Lebeau, L.; El Moualij, B.; Zorzi, W.; Coumans, B.; Grisar, T.; Bettendorff, L.; Int. J. Biochem. Cell Biol. 36, 1348-1364 (2004)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
expression in Escherichia coli, the recombinant enzyme is completely functional
Homo sapiens
Inhibitors
Inhibitors
Commentary
Organism
Structure
([2-[3-(4-Amino-2-methyl-pyrimidin-5-ylmethyl)-4-methyl-thiazol-5-yl]-ethoxy]-phosphonomethyl-phosphinoylmethyl)-phosphonic acid
;
Homo sapiens
ATP
very poor inhibitor
Homo sapiens
Ca2+
inhibits by competition with Mg2+
Homo sapiens
SDS
IC50: about 0.3% W/v. The inhibition is partially reversible, probably due to correct refolding of the denatured enzyme
Homo sapiens
thiamine triphosphate
-
Homo sapiens
Zn2+
inhibits at micromolar concentrations at pH 8.0, IC50: about 0.015-0.02 mM. Activates at pH 6.0
Homo sapiens
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.154
-
thiamine triphosphate
untagged recombinant enzyme
Homo sapiens
3.7
-
ATP
in presence of 8 mM ATP
Homo sapiens
Localization
Localization
Commentary
Organism
GeneOntology No.
Textmining
soluble
-
Homo sapiens
-
-
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Mg2+
required
Homo sapiens
Zn2+
inhibits at micromolar concentrations at pH 8.0, activates at pH 6.0
Homo sapiens
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Homo sapiens
-
-
-
Purification (Commentary)
Commentary
Organism
-
Homo sapiens
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
140
-
purified recombinant enzyme
Homo sapiens
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + H2O
the catalytic efficiency is about 4 orders of magnitude lower than for thiamine triphosphate
655738
Homo sapiens
ADP + phosphate
-
-
-
?
thiamine triphosphate + H2O
-
655738
Homo sapiens
thiamine diphosphate + phosphate
-
-
-
?
Temperature Stability [°C]
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
55
60
30 min, stable
Homo sapiens
68
-
30 min, 50% loss of activity
Homo sapiens
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.24
-
ATP
in presence of 8 mM Mg2+
Homo sapiens
140
-
thiamine triphosphate
-
Homo sapiens
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8.5
-
-
Homo sapiens
pH Range
pH Minimum
pH Maximum
Commentary
Organism
7
9.5
pH 7.0: about 60% of maximal activity, pH 9.5: about 75% of maximal activity
Homo sapiens
Ki Value [mM]
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.95
-
thiamine triphosphate
-
Homo sapiens
1.2
-
([2-[3-(4-Amino-2-methyl-pyrimidin-5-ylmethyl)-4-methyl-thiazol-5-yl]-ethoxy]-phosphonomethyl-phosphinoylmethyl)-phosphonic acid
-
Homo sapiens
IC50 Value
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.015
0.02
inhibits at micromolar concentrations at pH 8.0, IC50: about 0.015-0.02 mM. Activates at pH 6.0
Homo sapiens
Zn2+
Cloned(Commentary) (protein specific)
Commentary
Organism
expression in Escherichia coli, the recombinant enzyme is completely functional
Homo sapiens
IC50 Value (protein specific)
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.015
0.02
inhibits at micromolar concentrations at pH 8.0, IC50: about 0.015-0.02 mM. Activates at pH 6.0
Homo sapiens
Zn2+
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
([2-[3-(4-Amino-2-methyl-pyrimidin-5-ylmethyl)-4-methyl-thiazol-5-yl]-ethoxy]-phosphonomethyl-phosphinoylmethyl)-phosphonic acid
-
Homo sapiens
ATP
very poor inhibitor
Homo sapiens
Ca2+
inhibits by competition with Mg2+
Homo sapiens
SDS
IC50: about 0.3% W/v. The inhibition is partially reversible, probably due to correct refolding of the denatured enzyme
Homo sapiens
thiamine triphosphate
-
Homo sapiens
Zn2+
inhibits at micromolar concentrations at pH 8.0, IC50: about 0.015-0.02 mM. Activates at pH 6.0
Homo sapiens
Ki Value [mM] (protein specific)
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.95
-
thiamine triphosphate
-
Homo sapiens
1.2
-
([2-[3-(4-Amino-2-methyl-pyrimidin-5-ylmethyl)-4-methyl-thiazol-5-yl]-ethoxy]-phosphonomethyl-phosphinoylmethyl)-phosphonic acid
-
Homo sapiens
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.154
-
thiamine triphosphate
untagged recombinant enzyme
Homo sapiens
3.7
-
ATP
in presence of 8 mM ATP
Homo sapiens
Localization (protein specific)
Localization
Commentary
Organism
GeneOntology No.
Textmining
soluble
-
Homo sapiens
-
-
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Mg2+
required
Homo sapiens
Zn2+
inhibits at micromolar concentrations at pH 8.0, activates at pH 6.0
Homo sapiens
Purification (Commentary) (protein specific)
Commentary
Organism
-
Homo sapiens
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
140
-
purified recombinant enzyme
Homo sapiens
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + H2O
the catalytic efficiency is about 4 orders of magnitude lower than for thiamine triphosphate
655738
Homo sapiens
ADP + phosphate
-
-
-
?
thiamine triphosphate + H2O
-
655738
Homo sapiens
thiamine diphosphate + phosphate
-
-
-
?
Temperature Stability [°C] (protein specific)
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
55
60
30 min, stable
Homo sapiens
68
-
30 min, 50% loss of activity
Homo sapiens
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.24
-
ATP
in presence of 8 mM Mg2+
Homo sapiens
140
-
thiamine triphosphate
-
Homo sapiens
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8.5
-
-
Homo sapiens
pH Range (protein specific)
pH Minimum
pH Maximum
Commentary
Organism
7
9.5
pH 7.0: about 60% of maximal activity, pH 9.5: about 75% of maximal activity
Homo sapiens
Other publictions for EC 3.6.1.28
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
733393
Delvaux
Structural determinants of spe ...
Homo sapiens, Mus musculus
Biochim. Biophys. Acta
1830
4513-4523
2013
-
-
2
1
9
-
-
10
-
1
-
-
-
2
-
-
-
-
-
-
-
-
1
-
-
-
-
9
-
-
-
-
-
-
-
-
-
2
-
1
9
-
-
-
-
10
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
9
-
-
-
-
-
-
-
-
7
7
687693
Song
Structural basis for the catal ...
Mus musculus
J. Biol. Chem.
283
10939-10948
2008
-
-
-
-
-
-
1
1
1
1
1
1
-
2
-
-
-
1
-
-
-
-
2
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
1
1
1
1
-
-
-
-
-
-
-
-
2
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
696404
Kovacevic
The iron-regulated metastasis ...
Homo sapiens, Rattus norvegicus
Biochim. Biophys. Acta
1783
1981-1992
2008
2
-
2
-
2
-
-
-
-
-
-
2
-
2
-
-
-
-
-
3
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
2
-
2
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
3
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
667797
Szyniarowski Piot
Pig tissues express a catalyti ...
Homo sapiens, Sus scrofa
Biochim. Biophys. Acta
1725
93-102
2005
-
-
2
-
4
-
2
3
-
5
2
-
-
6
-
-
2
-
-
1
16
-
2
-
-
-
1
-
5
-
-
-
2
-
1
-
-
2
-
-
4
-
1
2
2
3
-
5
2
-
-
-
-
2
-
1
16
-
2
-
-
-
1
-
5
-
-
-
-
-
-
-
-
-
655738
Lakaye
Human recombinant thiamine tri ...
Homo sapiens
Int. J. Biochem. Cell Biol.
36
1348-1364
2004
-
-
1
-
-
-
6
2
1
2
-
-
-
3
-
-
1
-
-
-
1
-
2
-
-
-
2
2
1
1
-
-
2
-
1
-
-
1
-
-
-
-
1
7
2
2
1
2
-
-
-
-
-
1
-
-
1
-
2
-
-
-
2
2
1
1
-
-
-
-
-
-
-
-
656873
Czerniecki
Neuronal localization of the 2 ...
Rattus norvegicus
Neuroscience
125
833-840
2004
-
-
-
-
-
-
-
-
1
-
-
1
-
4
-
-
-
-
-
3
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
3
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
668963
Lakaye
Expression of 25 kDa thiamine ...
Mus musculus, Rattus norvegicus
Int. J. Biochem. Cell Biol.
36
2032-2041
2004
-
-
-
-
-
-
-
1
-
-
-
-
-
5
-
-
-
-
-
16
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
16
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
655198
Makarchikov
Thiamine triphosphate and thia ...
Bos taurus, Gallus gallus, Homo sapiens, quail, Rattus norvegicus, Sus scrofa
Cell. Mol. Life Sci.
60
1477-1488
2003
-
-
-
-
-
-
-
15
-
-
17
-
-
12
-
-
-
-
-
25
22
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15
-
-
17
-
-
-
-
-
-
25
22
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
656042
Lakaye
Molecular characterization of ...
Bos taurus, Homo sapiens
J. Biol. Chem.
277
13771-13777
2002
-
-
1
-
-
-
4
2
1
-
6
-
-
7
-
-
2
-
-
9
2
-
4
2
-
-
-
2
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
4
-
2
1
-
6
-
-
-
-
2
-
9
2
-
4
2
-
-
-
2
-
-
-
-
-
-
-
-
-
-
210023
Makarchikov
Thiamine triphosphatase activi ...
Bos taurus
Biochem. Mol. Biol. Int.
46
115-123
1998
-
-
-
-
-
-
-
2
-
-
1
-
-
2
-
-
-
-
-
1
-
-
2
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
1
-
-
2
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
210021
Bettendorff
Thiamine, thiamine phosphates, ...
Homo sapiens
J. Neurochem.
66
250-258
1996
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
-
-
-
-
1
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
210020
Makarchikov
Purification and characterizat ...
Bos taurus
Biochim. Biophys. Acta
1117
326-332
1992
-
-
-
-
-
1
-
1
1
6
2
-
-
2
-
-
1
-
-
2
1
2
7
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
1
6
2
-
-
-
-
1
-
2
1
2
7
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
210012
Matsuda
Membrane-associated thiamine t ...
Rattus norvegicus
Int. J. Biochem.
23
1111-1114
1991
-
-
-
-
-
-
9
-
1
-
-
-
-
2
-
-
-
-
-
2
-
-
3
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9
-
-
1
-
-
-
-
-
-
-
-
2
-
-
3
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
210022
Bettendorff
Solubilization and thiamine tr ...
Electrophorus electricus
Biochim. Biophys. Acta
1073
69-76
1991
4
-
-
-
-
-
3
-
1
-
-
-
-
2
-
-
-
-
-
2
-
1
1
-
-
-
1
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
3
-
-
1
-
-
-
-
-
-
-
-
2
-
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
210024
Bettendorff
Application of high-performanc ...
Electrophorus electricus
J. Chromatogr.
566
397-408
1991
-
-
-
-
-
-
-
-
-
1
-
1
-
3
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210019
Nishimune
Hydrolysis and synthesis of th ...
Aneurinibacillus aneurinilyticus, Escherichia coli, Escherichia coli W 1485
J. Nutr. Sci. Vitaminol.
33
113-127
1987
-
-
-
-
-
-
3
2
2
1
-
-
-
3
-
-
-
-
-
-
-
-
3
-
2
-
-
-
2
-
-
-
1
-
-
-
-
-
-
-
-
-
-
3
1
2
2
1
-
-
-
-
-
-
-
-
-
-
3
-
2
-
-
-
2
-
-
-
-
-
-
-
-
-
210018
Ogawa
Recent findings on ultracytoch ...
Rattus norvegicus
Ann. N. Y. Acad. Sci.
378
188-214
1982
-
-
-
-
-
-
-
-
4
-
-
1
-
1
-
-
-
-
-
3
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
1
-
-
-
-
-
3
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210017
Penttinen
The relation of the soluble th ...
Rattus norvegicus
Med. Biol.
59
177-184
1981
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
2
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210013
Barchi
Membrane-associated thiamin tr ...
Rattus norvegicus
J. Biol. Chem.
251
193-197
1976
-
-
-
-
-
-
1
-
1
3
-
-
-
1
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
3
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210014
Barchi
Membrane thiamine triphosphata ...
Rattus norvegicus
J. Neurochem.
26
715-720
1976
-
-
-
-
-
-
4
-
1
-
-
-
-
2
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
1
-
-
-
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210011
Iwata
Properties of thiamine di- and ...
Rattus norvegicus
J. Neurochem.
24
1209-1213
1975
-
-
-
-
-
-
4
-
2
-
-
-
-
1
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
2
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210009
Iwata
Role of thiamine metabolism in ...
Rattus norvegicus
Jpn. J. Pharmacol.
24
825-829
1974
-
-
-
-
-
-
2
-
1
-
-
-
-
2
-
-
-
-
-
3
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
-
-
3
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210010
Iwata
Role of thiamine metabolism in ...
Rattus norvegicus
Jpn. J. Pharmacol.
24
817-823
1974
-
-
-
-
-
-
1
-
3
2
-
-
-
2
-
-
-
-
-
3
-
-
4
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
3
2
-
-
-
-
-
-
-
3
-
-
4
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
210015
Iwata
-
Some properties of the enzyme ...
Rattus norvegicus
Thiamine (Proc. Pap. U. S. -Jpn. Semin, 2. Meeting)
2. Meeting
213-221
1974
-
-
-
-
-
-
4
-
2
2
-
-
-
1
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
2
2
-
-
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210016
Barchi
-
Thiamine triphosphatases in br ...
Rattus norvegicus
Thiamine (Proc. Pap. U. S. -Jpn. Semin, 2. Meeting)
195-212
1974
-
-
-
-
-
-
5
2
-
3
-
-
-
1
-
-
-
-
-
1
-
-
1
-
1
-
1
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
2
-
3
-
-
-
-
-
-
-
1
-
-
1
-
1
-
1
-
2
-
-
-
-
-
-
-
-
-
210006
Barchi
A membrane-associated thiamine ...
Rattus norvegicus
J. Biol. Chem.
247
7668-7673
1972
-
-
-
-
-
-
3
3
1
8
1
-
-
2
-
-
1
-
-
8
-
-
1
-
-
-
1
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
3
1
8
1
-
-
-
-
1
-
8
-
-
1
-
-
-
1
-
2
2
-
-
-
-
-
-
-
-
210007
Hashitani
The partial purification of th ...
Rattus norvegicus
J. Biol. Chem.
247
2117-2119
1972
-
-
-
-
-
-
4
1
1
-
-
-
-
2
-
-
-
-
-
6
1
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
1
1
-
-
-
-
-
-
-
-
6
1
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
210008
Barchi
Thiamine triphosphatase in the ...
Rattus norvegicus
Biochim. Biophys. Acta
255
402-405
1972
1
-
-
-
-
-
1
-
2
-
-
-
-
1
-
-
-
-
-
1
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
1
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-