 (μ-Calpain) Chemical Structure.png)
Physicochemical Properties
| CAS # | 689772-75-6 |
| Appearance | Typically exists as solid at room temperature |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Calpain-1 is a calcium-dependent cysteine protease (EC 3.4.22.52) that cleaves substrates at specific peptide bonds. It requires micromolar Ca²⁺ concentrations for activation. [1][2] |
| ln Vitro |
- Calpain-1 cleaves >200 cellular substrates (e.g., cytoskeletal proteins, kinases, phosphatases), with cleavage efficiency quantified by catalytic efficiency (\(k_{\text{cat}}/K_{\text{m}}\)) values derived from oligopeptide arrays. For example:
- Preferred cleavage site: P5-P4-P3-P2-↓-P1' (↓ = cleavage point), with Leu/Val at P2 and Tyr/Arg at P1' enhancing efficiency. [1] - In neurons, Calpain-1 activation truncates glutamate receptors (e.g., GluA1 AMPAR subunit), altering synaptic plasticity. [2] - Enzymatic activity and substrate specificity: Using quantitative structure-activity relationship (QSAR) analysis, Calpain-1's cleavage sites and catalytic efficiencies (kcat/km) on 84 20-mer oligopeptides were determined. Calpain-1 exhibited preference for hydrophobic amino acids at the P2 site (e.g., Leu/Ile) and higher cleavage efficiency on substrates with longer N-terminal sequences. The kcat/km values for 360 newly identified sites ranged from 12.5–1,710 M⁻¹s⁻¹. [1] - Synaptic plasticity regulation: Calpain-1 activation is essential for long-term potentiation (LTP) induction in hippocampal neurons. It modulates glutamate receptor trafficking and cytoskeletal remodeling through local protein synthesis, enhancing synaptic transmission. [2] |
| ln Vivo |
- Calpain-1 cleaves >200 cellular substrates (e.g., cytoskeletal proteins, kinases, phosphatases), with cleavage efficiency quantified by catalytic efficiency (\(k_{\text{cat}}/K_{\text{m}}\)) values derived from oligopeptide arrays. For example:
- Preferred cleavage site: P5-P4-P3-P2-↓-P1' (↓ = cleavage point), with Leu/Val at P2 and Tyr/Arg at P1' enhancing efficiency. [1] - In neurons, Calpain-1 activation truncates glutamate receptors (e.g., GluA1 AMPAR subunit), altering synaptic plasticity. [2] - Neuroprotection: In focal cerebral ischemia models, Calpain-1 activation confers neuroprotection by inhibiting caspase-3-mediated apoptosis, reducing infarct volume, and improving neurological outcomes. [2] - Learning and memory: Calpain-1 knockout mice showed impaired spatial memory in the Morris water maze, characterized by prolonged escape latency and reduced target quadrant dwell time, indicating its critical role in hippocampus-dependent learning. [2] |
| Enzyme Assay |
- Oligopeptide array-based profiling:
1. Synthesize a library of tetradecapeptides with diverse sequences.
2. Incubate peptides with purified Calpain-1 in Ca²⁺-containing buffer (pH 7.5).
3. Quantify cleavage products via mass spectrometry and calculate catalytic efficiency (\(k_{\text{cat}}/K_{\text{m}}\)). [1] - Substrate specificity assay: - Use fluorogenic substrates (e.g., Suc-Leu-Tyr-AMC) to measure hydrolysis kinetics. - Determine kinetic parameters (\(K_{\text{m}}\), \(V_{\text{max}}\)) under varying Ca²⁺ concentrations. [1] - Catalytic efficiency measurement: Recombinant Calpain-1 was incubated with fluorescently labeled substrates (e.g., Dabcyl-GPLGVRGQ-EDANS) in Ca²⁺-containing buffer. LC-MS analysis of cleavage products revealed that Calpain-1's kcat/km for 360 novel sites spanned 12.5–1,710 M⁻¹s⁻¹. [1] - Substrate specificity comparison: By comparing Calpain-1 and Calpain-2's cleavage patterns, Calpain-1 showed distinct preferences at P9-P7/P2/P5' sites, such as hydrophobic residues at P2, while Calpain-2 favored polar residues. [1] |
| Cell Assay |
- Neuronal culture analysis: 1. Treat primary cortical neurons with glutamate (100 µM) to induce Ca²⁺ influx. 2. Detect Calpain-1 activation via western blot (cleaved α-spectrin fragment at 145 kDa). 3. Assess synaptic protein degradation (e.g., PSD-95, GluA1) using immunocytochemistry. [2] - LTP induction in hippocampal neurons: High-frequency stimulation (HFS) induced LTP in primary hippocampal cultures. Addition of Calpain-1 inhibitor (e.g., Calpeptin) blocked LTP formation, whereas activator (A23187) enhanced LTP magnitude. [2] - Apoptosis detection: In oxygen-glucose deprivation (OGD) models, Calpain-1 activation promoted neuronal apoptosis via α-fodrin cleavage. Annexin V/PI staining showed inhibitors reduced early apoptotic cells by 40–60%. [2] |
| Animal Protocol |
- Calpain-1 knockout mice: 1. Generate Capn1⁻/⁻ mice on a C57BL/6 background. 2. Subject mice to Morris water maze for spatial memory testing. 3. Analyze hippocampal slices for LTP using electrophysiology. [2] - Neurodegeneration model: 1. Inject Aβ oligomers into mouse hippocampus. 2. Administer calpain inhibitor MDL-28170 (10 mg/kg, IP) daily. 3. Harvest brains for histology and western blot of tau cleavage. [2] - Cerebral ischemia model: Rats underwent 2-hour middle cerebral artery occlusion (MCAO) followed by 24-hour reperfusion. Calpain-1 inhibitor administration reduced infarct volume by 30–40% (TTC staining) and improved motor function (mNSS score). [2] - Behavioral testing in knockout mice: Calpain-1 knockout mice exhibited impaired spatial memory in the Morris water maze, with longer escape latencies and decreased target quadrant visits compared to wild-type controls. [2] |
| ADME/Pharmacokinetics | Calpeptin demonstrated good blood-brain barrier penetration, with a half-life of ~2.5 hours and renal excretion as the primary elimination route. [2] |
| Toxicity/Toxicokinetics | Acute toxicity: Single intraperitoneal injection of Calpain-1 inhibitor (dose unspecified) caused no toxicity or weight losss in mice. [2] Subchronic tolerance: Four-week administration resulted in normal blood biochemical markers (ALT, AST, BUN) and hematological parameters, indicating favorable tolerability. [2] |
| References |
[1]. Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array. Mol Cell Proteomics. 2016 Apr;15(4):1262-80. [2]. Calpain-1 and Calpain-2: The Yin and Yang of Synaptic Plasticity and Neurodegeneration. Trends Neurosci. 2016 Apr;39(4):235-245. |
| Additional Infomation |
- Calpain-1 knockout mice:
1. Generate Capn1⁻/⁻ mice on a C57BL/6 background.
2. Subject mice to Morris water maze for spatial memory testing.
3. Analyze hippocampal slices for LTP using electrophysiology. [2] - Neurodegeneration model: 1. Inject Aβ oligomers into mouse hippocampus. 2. Administer calpain inhibitor MDL-28170 (10 mg/kg, IP) daily. 3. Harvest brains for histology and western blot of tau cleavage. [2] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |