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 8 (FLICE, Mch5 and MACH) is an upstream caspase, which can activate all other caspases. Caspase 8 becomes activated through its interaction with the adaptor protein FADD. FADD binds caspase 8 when the cytokine receptor CD95 is activated. Caspase 8 is constitutively expressed as a 55 kD protein and, when activated, generates 18 and 17 Kd mature polypetides. Mice deficient in caspase 8 die in uterus due to impaired heart muscle development and congested accumulation of erythrocytes. Substrates of caspase 8 include caspase 3, 7 and 13. 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 8

Uniprot Number:

Q14790

Gene Name:

CASP8

Gene ID:

841

Gene Aliases:

MCH5|MACH|FLICE|Casp-8

Accession Number:

NM_001228

Organism:

Human

Construct Details:

Fragment (217-end), 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 8 expressed in E. coli is used. Test compound and/or vehicle is preincubated with 340 ng/ml enzyme‡ in modified HEPES buffer pH 7.4 for 15 minutes at 37⁰C. The reaction is initiated by addition of 50 μM Ac-IEVD-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-IETD-AMC

Incubation:

60 min at 37°C

Control Inhibitor:

Ac-IETD-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

0.011μM - IC50

PubChem ID

5311139

Name

Ac-YVAD-CHO

Measurement

1.9μM - IC50

PubChem ID

11225800

Name

Ac-LEVD-CHO

Measurement

0.0033μM - IC50

PubChem ID

15487887

Name

Ac-VEID-CHO

Measurement

0.026μM - IC50

PubChem ID

25108684

Name

Z-VDVAD-FMK

Measurement

0.14μM - IC50

PubChem ID

44135117

Name

Ac-IETD-CHO

Measurement

0.004μM - IC50

PubChem ID

122173007

Name

Z-LEHD-fmk

Measurement

0.08μM - IC50

PubChem ID

Name

Z-WEHD-FMK

Measurement

0.22μM - IC50

Clinical Relevance

Therapeutic Area:

Oncology/Immuno-Oncology

Additional Information

Brand:

LeadHunter

Testing Location:

Taiwan - Taipei

RELATED SOLUTIONS

Item: PP318
Item: PP325