Background
Leupeptin, Microbial is a broad-spectrum serine and cysteine protease inhibitor [1]. Leupeptin, Microbial is a reversible protease inhibitor, It acts on bovine trypsin, human plasminase, bovine splenic cathepsin B, and recombinant human caltrypsin with Ki values of 35 nM, 3.4 μM, 6 nM, and 72 nM[7].
The protease inhibitor Leupeptin, Microbial prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells [3]. Leupeptin, Microbial-mediated inhibition of trypsin-like proteases maintains substrate mycelium development, whereas proteolytic degradation of Leupeptin, Microbial in stationary phase cultures derepresses the trypsin-like proteases, leading to the digestion of substrate mycelium and promotion of aerial mycelium formation [2]. As the calpain inhibitor Leupeptin, Microbial primarily protected hair cells from neomycin[5]. The expression of hepatitis B surface antigen (HBsAg) from Leupeptin recovered up to 50% of the cell suspension culture [6].
Leupeptin, Microbial was well tolerated in mice. Leupeptin, Microbial significantly increased LC3b-II in a dose-dependent way in the total tissue extract and lysosomal enrichment portion (LE portion). At the level of electron microscopy (EM), Leupeptin, Microbial induced the accumulation of electron-dense vesicle structures [4].
参考文献:
[1]. Aoyagi T, Miyata S, et,al. Biological activities of leupeptins. J Antibiot (Tokyo). 1969 Nov;22(11):558-68. doi: 10.7164/antibiotics.22.558. PMID: 4243683.
[2]. Kim IS, Kim YB, et,al. Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin. Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):539-45. doi: 10.1042/bj3310539. PMID: 9531495; PMCID: PMC1219386.
[3]. Appleyard G, Tisdale M. Inhibition of the growth of human coronavirus 229E by leupeptin. J Gen Virol. 1985 Feb;66 ( Pt 2):363-6. doi: 10.1099/0022-1317-66-2-363. PMID: 3968542.
[4]. Haspel J, Shaik RS, et,al. Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy. 2011 Jun;7(6):629-42. doi: 10.4161/auto.7.6.15100. Epub 2011 Jun 1. PMID: 21460622; PMCID: PMC3127049.
[5]. Aoyagi T, Miyata S, et,al. Biological activities of leupeptins. J Antibiot (Tokyo). 1969 Nov;22(11):558-68. doi: 10.7164/antibiotics.22.558. PMID: 4243683.
[6]. Coffin AB, Williamson KL, et,al. Profiling drug-induced cell death pathways in the zebrafish lateral line. Apoptosis. 2013 Apr;18(4):393-408. doi: 10.1007/s10495-013-0816-8. PMID: 23413197; PMCID: PMC3627356.
[7]. Ganapathi TR, Sunil Kumar GB, et,al. Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures. Plant Cell Rep. 2007 Sep;26(9):1575-84. doi: 10.1007/s00299-007-0379-7. Epub 2007 May 30. PMID: 17534624.
Leupeptin, Microbial 是一种广谱丝氨酸和半胱氨酸蛋白酶抑制剂[1]。 Leupeptin, Microbial 是一种可逆的蛋白酶抑制剂，它作用于牛胰蛋白酶、人纤溶酶、牛脾脏组织蛋白酶 B 和重组人钙蛋白酶，Ki 值为 35 nM、3.4 μM、6 nM 和 72 nM[7]< /sup>.
蛋白酶抑制剂 Leupeptin Microbial 阻止了 MRC-C 细胞培养物中人类冠状病毒株 229E 的增殖[3]。亮肽素，微生物介导的胰蛋白酶样蛋白酶抑制维持底物菌丝体发育，而亮肽素的蛋白水解降解，微生物在稳定期培养物中去抑制胰蛋白酶样蛋白酶，导致底物菌丝体的消化和气生菌丝体形成的促进 [2].作为钙蛋白酶抑制剂 Leupeptin，Microbial 主要保护毛细胞免受新霉素的侵害[5]。 Leupeptin的乙型肝炎表面抗原（HBsAg）的表达在细胞悬浮培养物中恢复了50%[6]。
Leupeptin, Microbial 在小鼠中具有良好的耐受性。 Leupeptin, Microbial 在总组织提取物和溶酶体富集部分（LE 部分）中以剂量依赖性方式显着增加 LC3b-II。在电子显微镜（EM）水平，亮肽素、微生物诱导电子致密囊泡结构的积累[4]。
Protocol
| Leupeptin analysis [1]: |
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Preparation Method
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The concentration of Leupeptin, Microbial was calculated by enzyme inhibition assay using 50 pg of papain and 0.2 mM of N-benzoylL-arginine ethyl ester as the target protease and papain substrate, respectively. Enzyme reactions were carried out at 25 ℃ and pH 6.2.
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Applications
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Leupeptin, Microbial is a leucine-specific protease and a metalloprotease.
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| Cell experiment [2]: |
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Cell lines
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MRC-C cell
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Preparation Method
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To investigate the effect of Leupeptin, Microbial on virus yield, MRC-C cultures in 35 mm dishes were infected by adsorption of virus from 0T ml concentrated suspension of HCV 229E for 1 h at 36 ℃. The cultures were washed twice and overlaid with 2 ml Eagle’s MEM plus 0.2% bovine serum albumin and 20 mm-HEPES buffer pH 7.4. Leupeptin, Microbial at a range of concentrations was added to the virus inoculum, to the maintenance medium and also to maintenance medium used to treat the cultures for 1 h before infection. After 24 h at 36 ℃ in air, the cells were scraped into the medium and disrupted by sonication.
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Reaction Conditions
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0.4 to l00 μg/ml for 25 h（1 h before infection to 24 h after infection）
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Applications
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The protease inhibitor Leupeptin, Microbial prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells.
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| Animal experiment [3]: |
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Animal models
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C57BL/6NCrl male mouse
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Preparation Method
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Mice received i.p. injections of 0.5 ml sterile Phosphate Buffered Saline or 0.5 ml PBS containing 9–40 mg/kg Leupeptin, Microbial hemisulfate.
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Dosage form
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0, 9, 18 36 and 40 mg/kg Leupeptin, Microbial; Intraperitoneal injection
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Applications
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Rate of LC3b accumulation after Leupeptin, Microbial treatment was greatest in the liver and lowest in spleen.
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参考文献:
[1]. Kim IS, Kim YB, et,al. Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin. Biochem J. 1998 Apr 15;331 (Pt 2)(Pt 2):539-45. doi: 10.1042/bj3310539. PMID: 9531495; PMCID: PMC1219386.
[2]. Appleyard G, Tisdale M. Inhibition of the growth of human coronavirus 229E by leupeptin. J Gen Virol. 1985 Feb;66 (Pt 2):363-6. doi: 10.1099/0022-1317-66-2-363. PMID: 3968542.
[3]. Haspel J, Shaik RS, et,al. Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy. 2011 Jun;7(6):629-42. doi: 10.4161/auto.7.6.15100. Epub 2011 Jun 1. PMID: 21460622; PMCID: PMC3127049.
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