Information on EC 3.4.17.12 - carboxypeptidase M

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.17.12
-
RECOMMENDED NAME
GeneOntology No.
carboxypeptidase M
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cleavage of C-terminal arginine or lysine residues from polypeptides
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
120038-28-0
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
in HEK-293 cells stably transfected with kinin B1 receptor, co-expression of carboxypeptidase M enhances des-Arg10-kallidin-stimulated increases in intracellular Ca2+ or phosphoinositide turnover. Carboxypeptidase M increases kinin B1 receptor affinity for des-Arg10-kallidin by 5-fold but has no effect on basal kinin B1 receptor-dependent phosphoinositide turnover. Soluble, recombinant carboxypeptidase M binds to HEK cells expressing kinin B1 receptor without stimulating receptor signaling. Carboxypeptidase M positive allosteric action is independent of enzyme activity but depended on interaction of its C-terminal domain with the kinin B1 receptor extracellular loop 2. Disruption of the carboxypeptidase M/kinin B1 receptor interaction or knockdown of carboxypeptidase M in cytokine-treated primary human endothelial cells inhibits the allosteric enhancement of carboxypeptidase M on kinin B1 receptor des-Arg10-kallidin binding or ERK1/2 activation. Carboxypeptidase M also enhances the des-Arg10-kallidin-induced kinin B1 receptor conformational changes
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Ala-Ser-His-Leu-Gly-Leu-Ala-Arg + H2O
Ala-Ser-His-Leu-Gly-Leu-Ala + Arg
show the reaction diagram
-
human anaphylatoxin C3A fragment 70-77
-
-
?
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg + H2O
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe + arginine
show the reaction diagram
benzoyl-Ala-Arg + H2O
benzoyl-Ala + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Ala-Lys + H2O
benzoyl-Ala + Lys
show the reaction diagram
-
-
-
?
benzoyl-Asn-Arg + H2O
benzoyl-Asn + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Gln-Arg + H2O
benzoyl-Gln + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Gly-Arg + H2O
benzoyl-Gly + Arg
show the reaction diagram
benzoyl-Gly-argininic acid + H2O
?
show the reaction diagram
-
-
-
-
?
benzoyl-Gly-argininic acid + H2O
benzoyl-Gly + argininic acid
show the reaction diagram
-
-
-
-
?
benzoyl-Gly-L-Arg + H2O
benzoyl-Gly + L-Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Gly-Lys + H2O
benzoyl-Gly + Lys
show the reaction diagram
benzoyl-His-Arg + H2O
benzoyl-His + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Ile-Arg + H2O
benzoyl-Ile + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-L-Asn-L-Arg + H2O
benzoyl-L-Asn + L-Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Leu-Arg + H2O
benzoyl-Leu + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Met-Arg + H2O
benzoyl-Met + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Phe-Arg + H2O
benzoyl-Phe + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Ser-Arg + H2O
benzoyl-Ser + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Thr-Arg + H2O
benzoyl-Thr + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Trp-Arg + H2O
benzoyl-Trp + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Tyr-Arg + H2O
benzoyl-Tyr + Arg
show the reaction diagram
-
-
-
-
?
benzoyl-Val-Arg + H2O
benzoyl-Val + Arg
show the reaction diagram
-
-
-
-
?
Bradykinin + H2O
?
show the reaction diagram
bradykinin(-Phe-Arg) + H2O
?
show the reaction diagram
-
-
-
-
?
dansyl-Ala-Arg + H2O
?
show the reaction diagram
-
substrate for determination of CPM activity
-
-
?
Dansyl-Ala-Arg + H2O
Dansyl-Ala + Arg
show the reaction diagram
dansyl-L-Ala-L-Arg + H2O
dansyl-L-Ala + L-Arg
show the reaction diagram
dynorphin A(1-13) + H2O
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu + Lys
show the reaction diagram
-
i.e. Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys
-
-
?
EGF + H2O
des-Arg53-EGF + arginine
show the reaction diagram
-
EGF, epidermal growth factor
-
-
?
epidermal growth factor + H2O
dea-Arg53-epidermal growth factor + arginine
show the reaction diagram
-
-
-
-
?
furylacryloyl-Ala-Arg + H2O
furylacryloyl-Ala + Arg
show the reaction diagram
-
-
-
-
?
furylacryloyl-Ala-Lys + H2O
furylacryloyl-Ala + Lys
show the reaction diagram
-
-
-
-
?
furylacryloyl-L-Ala-L-Arg + H2O
furylacryloyl-L-Ala + L-Arg
show the reaction diagram
-
-
-
-
?
Gly-Gly-Arg + H2O
Gly-Gly + Arg
show the reaction diagram
-
-
-
-
?
Gly-Leu-Ala-Arg + H2O
Gly-Leu-Ala + Arg
show the reaction diagram
-
human anaphylatoxin C3A fragment 74-77
-
-
?
Hippuryl-L-Lys + H2O
Hippuric acid + L-Lys
show the reaction diagram
-
-
-
-
?
His-Leu-Gly-Leu-Ala-Arg + H2O
His-Leu-Gly-Leu-Ala + Arg
show the reaction diagram
-
human anaphylatoxin C3A fragment 72-77
-
-
?
Leu-Ala-Arg + H2O
Leu-Ala + Arg
show the reaction diagram
-
human anaphylatoxin C3A fragment 75-77
-
-
?
Leu-Gly-Leu-Ala-Arg + H2O
Leu-Gly-Leu-Ala + Arg
show the reaction diagram
-
human anaphylatoxin C3A fragment 73-77
-
-
?
Leu5-Arg6-enkephalin + H2O
?
show the reaction diagram
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg + H2O
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe + arginine
show the reaction diagram
Met5-Arg6-enkephalin + H2O
?
show the reaction diagram
Met5-Lys6-enkephalin + H2O
?
show the reaction diagram
N-(4-hydroxybenzoyl)-Gly-Arg + H2O
N-(4-hydroxybenzoyl)-Gly + L-arginine
show the reaction diagram
-
-
-
-
?
Pro-Gly-Lys-Ala-Arg + H2O
Pro-Gly-Lys-Ala + Arg
show the reaction diagram
-
-
-
-
?
SDF-1alpha + H2O
des-lys SDF-1alpha + lysine
show the reaction diagram
-
stromal cell-derived factor
-
-
?
Ser-His-Leu-Gly-Leu-Ala-Arg + H2O
Ser-His-Leu-Gly-Leu-Ala + Arg
show the reaction diagram
-
human anaphylatoxin C3A fragment 71-77
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg + H2O
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe + arginine
show the reaction diagram
-
bradykinin
-
-
?
EGF + H2O
des-Arg53-EGF + arginine
show the reaction diagram
-
EGF, epidermal growth factor
-
-
?
epidermal growth factor + H2O
dea-Arg53-epidermal growth factor + arginine
show the reaction diagram
-
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
-
addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297%
KNO3
-
addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297%
Na2SO4
-
addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297%
NaCl
-
addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297%
NaNO3
-
addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wilde-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297%
additional information
-
the enzyme is a metallopeptidase
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2-guanidinoethylmercapto)succinic acid
1,10-phenanthroline
1,4-dithiothreitol
-
-
2-Guanidinoethylmercaptosuccinic acid
-
strong CPM inhibitor
2-mercaptomethyl-3-guanidinoethyl thiopropanoic acid
2-mercaptomethyl-3-guanidinoethylthiopropanoic acid
-
-
bathophenanthroline disulfonic acid
-
-
Cadmium acetate
-
-
Cd(CH3COO)2
-
-
Cd2+
-
-
Co2+
-
-
DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid
DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid
DL-mercaptomethyl-3-guanidino-ethylthiopropanoic acid
-
-
EDTA
-
-
Guanidinoethyl mercaptosuccinic acid
Guanidinoethylmercaptosuccinic acid
-
-
MERGETPA
-
-
Zn2+
-
-
additional information
-
expression level of the gene carboxypeptidase M decreased after a Salmonella infection
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
G-CSF
-
granulocyte-colony stimulating factor
-
IFN-gamma
-
CPM activity can be upregulated approximately two-fold in HLMVECs by IL-1beta plus IFN-gamma treatment
-
IL-1beta
-
CPM activity can be upregulated approximately two-fold in HLMVECs by IL-1beta plus IFN-gamma treatment, IL-1beta alone can upregulate CPM expression
-
lipopolysaccharide
-
following stimulation with bacterial lipopolysaccharide a marginal upregulation of the expression of carboxypeptidase M is observed
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.033
Ala-Ser-His-Leu-Gly-Leu-Ala-Arg
-
CPMwt, pH 7.4, 37°C
0.115 - 0.18
benzoyl-Ala-Arg
0.343
benzoyl-Asn-Arg
-
CPMwt, pH 7.4, 37°C
0.127
benzoyl-Gln-Arg
-
CPMwt, pH 7.4, 37°C
0.255 - 0.29
benzoyl-Gly-Arg
0.046 - 0.049
benzoyl-Gly-argininic acid
10.15 - 10.65
Benzoyl-Gly-Lys
0.239
benzoyl-His-Arg
-
CPMwt, pH 7.4, 37°C
0.205
benzoyl-Ile-Arg
-
CPMwt, pH 7.4, 37°C
0.168
benzoyl-Leu-Arg
-
CPMwt, pH 7.4, 37°C
0.13 - 0.138
benzoyl-Met-Arg
0.0584
benzoyl-Phe-Arg
-
CPMwt, pH 7.4, 37°C
0.219
benzoyl-Ser-Arg
-
CPMwt, pH 7.4, 37°C
0.192
benzoyl-Thr-Arg
-
CPMwt, pH 7.4, 37°C
0.069
benzoyl-Trp-Arg
-
CPMwt, pH 7.4, 37°C
0.048
benzoyl-Tyr-Arg
-
CPMwt, pH 7.4, 37°C
0.264
benzoyl-Val-Arg
-
CPMwt, pH 7.4, 37°C
0.016
bradykinin
0.0277
bradykinin(-Phe-Arg)
-
CPMwt, pH 7.2, 37°C
0.059 - 0.28
Dansyl-Ala-Arg
0.068 - 0.09
furylacryloyl-Ala-Arg
1.152
furylacryloyl-Ala-Lys
-
CPMwt, pH 7.2, 37°C
1.181
Gly-Gly-Arg
-
CPMwt, pH 7.2, 37°C
0.05
Gly-Leu-Ala-Arg
-
CPMwt, pH 7.4, 37°C
0.028
His-Leu-Gly-Leu-Ala-Arg
-
CPMwt, pH 7.4, 37°C
0.027
Leu-Ala-Arg
-
CPMwt, pH 7.4, 37°C
0.041
Leu-Gly-Leu-Ala-Arg
-
CPMwt, pH 7.4, 37°C
0.063
Leu5-Arg6-enkephalin
-
-
0.046
Met5-Arg6-enkephalin
-
-
0.375
Met5-Lys6-enkephalin
-
-
0.206 - 0.241
N-(4-hydroxybenzoyl)-Gly-Arg
0.035
Pro-Gly-Lys-Ala-Arg
-
CPMwt, pH 7.2, 37°C
0.051
Ser-His-Leu-Gly-Leu-Ala-Arg
-
CPMwt, pH 7.4, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.4
Ala-Ser-His-Leu-Gly-Leu-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
77 - 107
benzoyl-Ala-Arg
35
benzoyl-Asn-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
22
benzoyl-Gln-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
3.9 - 6.07
benzoyl-Gly-Arg
273 - 316
benzoyl-Gly-argininic acid
9.6 - 11
Benzoyl-Gly-Lys
14
benzoyl-His-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
1
benzoyl-Ile-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
19
benzoyl-Leu-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
87 - 130
benzoyl-Met-Arg
33
benzoyl-Phe-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
60
benzoyl-Ser-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
22.9
benzoyl-Thr-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
23
benzoyl-Trp-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
27
benzoyl-Tyr-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
7.7
benzoyl-Val-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
2.45
bradykinin
30
bradykinin(-Phe-Arg)
Homo sapiens
-
CPMwt, pH 7.2, 37°C
0.233 - 17.6
Dansyl-Ala-Arg
32 - 40
furylacryloyl-Ala-Arg
11
furylacryloyl-Ala-Lys
Homo sapiens
-
CPMwt, pH 7.2, 37°C
1.9
Gly-Gly-Arg
Homo sapiens
-
CPMwt, pH 7.2, 37°C
8.2
Gly-Leu-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
6.6
His-Leu-Gly-Leu-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
4.2
Leu-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
6.1
Leu-Gly-Leu-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
1.77
Leu5-Arg6-enkephalin
Homo sapiens
-
-
15.6
Met5-Arg6-enkephalin
Homo sapiens
-
-
11.1
Met5-Lys6-enkephalin
Homo sapiens
-
-
4.9 - 5.7
N-(4-hydroxybenzoyl)-Gly-Arg
11
Pro-Gly-Lys-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.2, 37°C
6.7
Ser-His-Leu-Gly-Leu-Ala-Arg
Homo sapiens
-
CPMwt, pH 7.4, 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000003 - 0.000006
MERGETPA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.344
-
-
14
-
CPMwt with dansyl-Ala-Arg
19
-
hCPM with dansyl-Ala-Arg
additional information
-
16474, RFU min-1 mg-1, purification step Mono Q, RFU - relative fluorescence units; 2415 RFU min-1 mg-1, purification step Superdex 200 HR, RFU - relative fluorescence units; 9.4 RFU min-1 mg-1, purification step supernatant, RFU - relative fluorescence units; purified recombinant enzyme, relative fluorescent units
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
-
hydrolysisdansyl-Ala-Arg
6.5 - 7.5
-
-
7 - 8
-
-
7.2
-
enzyme assay
7.4
-
hearts are perfused with Krebs Henseleit solution at a rate of 10 ml per min, perfusate samples are analysed and bradykinin metabolites are characterized
additional information
-
neutral pH-optimum
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
pH 5.0: 20% of maximal activity, pH 8.0: 72% of maximal activity
5.5 - 8
-
pH 5.5: about 35% of maximal activity, pH 7.0-8.0: activity maximum
5.5 - 9
-
pH 5.5: 25% of maximal activity, pH 9.0: about 60% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
interstitial trophoblast
Manually annotated by BRENDA team
-
arterial endothelium
Manually annotated by BRENDA team
-
erythroid cell progenitor
Manually annotated by BRENDA team
-
very low activity
Manually annotated by BRENDA team
-
myeloid cell progenitor
Manually annotated by BRENDA team
-
megakaryocytic cell progenitor
Manually annotated by BRENDA team
-
peripheral
Manually annotated by BRENDA team
-
CPM mRNA expression is very low in the human pancreas
Manually annotated by BRENDA team
-
mobilized peripheral blood CD34+ cell
Manually annotated by BRENDA team
-
tumor cells of renal cell carcinoma subtypes lose carboxypeptidase M expression upon dedifferentiation. There is a correlation between low carboxypeptidase M expression and tumor cell type. Carboxypeptidase M staining is intense on phagocytotic tumor-associated macrophages, and enzyme is also detected in the tumor-associated vasculature. Coexistence of carboxypeptidase M and the epidermal growth factor receptor is detected in papillary renal cell carcinoma
Manually annotated by BRENDA team
-
very low activity
Manually annotated by BRENDA team
-
endovascular
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
-
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
47600
-
1 * 62000, glycosylated enzyme, SDS-PAGE, 1 * 47600, deglycosylated enzyme, SDS-PAGE
48000
-
deglycosylated protein, calculated from amino acid sequence
49000
-
determined by SDS-PAGE
54000
-
x * 54000, SDS-PAGE
65000
-
determined by SDS-PAGE and immunoblotting
73000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
-
catalytic domain structure
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified, recombinant, glycosylphosphatidylinositol-free enzyme, X-ray diffraction structure determination and analysis at 3.0 A resolution, modelling
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
1 h, room temperature, 33% loss of activity
647252
4.3
-
1 h, room temperature, 17% loss of activity
647252
4.5 - 5
-
1 h, room temperature, stable
647252
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
wild-type enzyme and mutant enzymes E260Q and E260A are stable
45
-
15 min, 50% inactivation of mutant enzyme E260A
49
-
15 min, 15 min, 50% inactivation of mutant enzyme E260Q
50
-
15 min, complete inactivation of mutant enzyme E260A
53
-
15 min, wild-type enzyme retains 50% of its activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 50% v/v glycerol, stable for at least 1 year
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme from placental microvillus membranes by solubilization through either phosphatidylinositol-specific phospholipase C or CHAPS, ion exchange and arginine affinity chromatography, and gel filtration
-
recombinant 3'-truncated, glycosylphosphatidylinositol-free, soluble enzyme from insect cells by anion exchange chromatography and gel filtration to homogeneity
-
recombinant enzyme 1753fold from Pichia pastoris strain GS115 by gel filtration and anion exchange chromatography to homogeneity; the CPM secreted in the supernatant is purified by a two-step procedure consisting of gel filtration and ion-exchange chromatography
-
recombinant proteins are purified by affinity chromatography, membrane extracts are prepared from rat lungs and loaded onto a heparin affinity column, followed by hydroxyapatite and cation exchange chromatographies
-
wild type carboxypeptidase M, CPMwt, is purified from Pichia pastoris supernatant using hydrophobic interaction chromatography and affinity chromatography, hCPM is purified from human prostasomes
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CPM cDNAs are cloned into pcDNA3 or pcDNA6 for expression in mammalian cells, to express N-terminal CFP- or YFP-tagged CPM pECFP-C1 and pEYFP-C1 vectors are used, to generate a fusion protein of CPM attached to the extracellular N-terminus of B1R pcDNA3 and 6 vectors are used
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DNA and amino acid sequence determination and analysis, genetic structure, localization on chromosome 12q13-q15, expression in insect cells via baculovirus transfection
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expressed in Escherichia coli strain DH5alpha
expressed in HEK-293 cells, in Pichia pastoris, and in baculovirus-infected insect cells
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expression in Pichia pastoris; PCR-amplified fragment encoding hCPM is cloned into the pGEM-T-Easy cloning vector, and subsequent into the pPIC9 vector for transfomation of Pichia pastoris cells
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expression of 3'-truncated, glycosylphosphatidylinositol-free, soluble enzyme in insect cells using the baculovirus transfection system
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expression of wild-type and mutant enzymes in COS-7 cells or HEK-293 cells. The wild-type and S406A and S406T mutants are expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, the S406P mutant is notz and is retained in a perinuclear location. Expression in baculovirus infected cells in a glycophosphatidyl-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidyl anchoring is secreted at high levels into the medium. Both forms have lower molecular masses than native placental enzyme indicating a minimal glycosylation
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into the pCMV5, pGex4T-1 and pMalC2 vector for expression in COS-7 and Escherichia coli cells
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placental cDNA is incorporated in the pICZalphaA shuttle vector, signal peptide and C-terminal hydrophobic membrane anchor signal are removed
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
oxidative stress reduction by epoetin delta goes along with the upregulation of renoprotective genes. The expression of carboxypeptidase M is significantly upregulated upon erythropoietin administration in and only in those cell cultures in which an erythropoietin-induced cytoprotective effect is seen. In cultures in which epoetin delta does not show protective effects, no upregulation of carboxypeptidase M is observed
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E260A
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mutation reduces the ratio of turnover-numver to Km-value by 104fold and further decreases stability.Addition of 0.5 M NaCl to the assay buffer does not significantly alter the activities of the wild-type enzyme with 0.2 mM dansyl-Ala-Arg or the E260Q mutant enzyme but does substantially increase the activity of the E260A mutant, 219%. The enhancement of E260A is not specific for NaCl, as similar increases are detected with other salts such as NaNO3, 249%, KNO3, 256%, KCl, 256%, or Na2SO4, 297%
E260Q
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mutation has minimal effects on kinetic parameters, but decreased heat stability
S406A
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very similar to the wild-type enzyme with regard to expression and release by phosphatidylinositol-specific phospholipase C
S406P
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the wild-type and S406A and S406T mutants are expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, the S406P mutant is not and is retained in a perinuclear location
S406T
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very similar to the wild-type enzyme with regard to expression and release by phosphatidylinositol-specific phospholipase C
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
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CPM, a gene that encodes carboxypeptidase M, is consistently amplified in liposarcomas but not in different subtypes of lipoma or normal fat, CPM could be used as an alternative and novel diagnostic tool for these tumors
medicine