Biological Information

Background Information:

Caspases are the executioners of programmed cell death (apoptosis), a normal process used physiologically to eliminate specific cells or cell types without damaging surrounding cells or causing an inflamatory response. All caspases belong to a family of proteases characterized by a cysteine residue in the active site and display specificity for Asp residues. Caspases are present in all cells as a procaspase, which becomes activated through proteolytic cleavage by other proteases including other caspases. Caspase-3 (CPP32, Yama, and apopain) is constitutively expressed in cells as a 32 kD procaspase. Activation of caspase 3 occurs by cleavage of the procaspase into 12 and 17 kD mature fragments by caspase 8, 6 and GranzymeB. Mice deficient in caspase 3 die within three weeks of birth due to failure of neuronal apoptosis. These mice display hyperplasia, duplicated brain structures and ectopic cell masses. However, the defects are restricted to the neural tissues, other organs are normal; indicating that caspase 3 has a non-redundant role in neural apoptosis. Caspase inhibitors are currently sought as therapeutics for such diseases as Alzheimer's, Parkinson's and cerebral/myocardial ischemia.

Family:

Protease

Sub Family:

Caspase

Class:

Cysteine Peptidase

Protein Name:

Caspase 3

Uniprot Number:

P42574

Gene Name:

CASP3

Gene ID:

836

Gene Aliases:

CPP32|CPP32B|Yama|apopain

Accession Number:

NM_004346

Organism:

Human

Construct Details:

Fragment, Recombinant

Assay Information

Assay Type:

Biochemical

Assay Sub Type:

Enzymatic

Functional Mode:

Antagonist

Detection Method:

Spectrofluorimetry

Measured Response:

Fluorescence

Testing Information

Procedure Summary:

Human recombinant caspase 3 expressed in E. coli is used. Test compound and/or vehicle is preincubated with 150 U/ml enzyme‡ in modified HEPES buffer pH 7.4 for 15 minutes at 37⁰C. The reaction is initiated by addition of 50 μM Ac-DEVD-AMC for another 60 minute incubation period. Determination of the amount of AMC formed is read spectrofluorimetrically at 360 nm/465 nm. Compounds are screened at 10 μM.

Substrate:

50 µM Ac-DEVD-AMC

Incubation:

60 min at 37°C

Control Inhibitor:

Ac-DEVD-CHO

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

2

Turnaround Time

Standard:

15 Business Days

Reference Compound Data

PubChem ID

644345

Name

Ac-DEVD-CHO

Measurement

0.0049μM - IC50

PubChem ID

5311139

Name

Ac-YVAD-CHO

Measurement

>10μM - IC50

PubChem ID

11225800

Name

Ac-LEVD-CHO

Measurement

0.057μM - IC50

PubChem ID

15487887

Name

Ac-VEID-CHO

Measurement

0.48μM - IC50

PubChem ID

25108684

Name

Z-VDVAD-FMK

Measurement

0.46μM - IC50

PubChem ID

44135117

Name

Ac-IETD-CHO

Measurement

0.18μM - IC50

PubChem ID

122173007

Name

Z-LEHD-fmk

Measurement

1.4μM - IC50

PubChem ID

Name

Z-WEHD-FMK

Measurement

5.4μM - IC50

Clinical Relevance

Therapeutic Area:

CNS/Addiction/Pain

Additional Information

Brand:

LeadHunter

Testing Location:

Taiwan - Taipei

RELATED SOLUTIONS

Item: PP318
Item: PP325