Bestatin

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, Sao Tome and Principe

Bestatin is a chemical compound used as a laboratory tool in research and development. It functions as an inhibitor of the enzyme aminopeptidase, which is involved in various biological processes. Bestatin is commonly utilized in experimental settings to explore the role of aminopeptidase in cellular and molecular mechanisms.

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Lab products found in correlation

Leupeptin, by Merck Group (41 mentions) Aprotinin, by Merck Group (35 mentions) Pepstatin a, by Merck Group (24 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (13 mentions) Pepstatin, by Merck Group (9 mentions) Protease inhibitor cocktail, by Merck Group (8 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (7 mentions) Amastatin, by Merck Group (6 mentions) Captopril, by Merck Group (5 mentions) 1 10 phenanthroline, by Merck Group (5 mentions) Chymostatin, by Merck Group (4 mentions) Bovine serum albumin (bsa), by Merck Group (4 mentions) Thiorphan, by Merck Group (4 mentions) Insulin, by Merck Group (3 mentions) Tw150f 3, by World Precision Instruments (3 mentions) Infinite 200, by Tecan (3 mentions) Z pro prolinal, by Enzo Life Sciences (3 mentions) Mca ala pro lys dnp oh, by Enzo Life Sciences (3 mentions) Penicillin, by Thermo Fisher Scientific (3 mentions) Naloxone, by Merck Group (3 mentions)

Spelling variants of this product

Bestatin hydrochloride (13 citations) Bestatin HCl (4 citations)

89 protocols using bestatin

Polyclonal Antibodies for Sorghum C4-PEPC

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Polyclonal antibodies against: (i) synthetic peptides corresponding to sorghum C₄-PEPC C-terminus [(Y) 942EDTLILTMKGIAAGMQNTG960] and dephosphorylated N-terminus [4ERHHSIDAQLRALAPGKVSEE24(YG)], C19-IgG and N24-IgG, respectively, were purchased from NEOSYSTEM S.A. (Strasbourg, France); and (ii) native C₄-PEPC from sorghum leaves (PEPC-IgG) achieved as described in Pacquit et al. (1995) (link).
Phospholipids with varying format and fatty acyl chain composition were all purchased from Avanti Polar Lipids, (Alabaster, AL, United States). Lipids in chloroform stock were dried with gaseous N₂, rehydrated in 0.1 M Tris–HCl buffer (pH 8) and sonicated prior to adding to the assays. Protease inhibitors used were: Chymostatin (cysteine protease, chymotrypsin and elastase inhibitor); phenylmethane sulfonyl fluoride (PMSF; serine protease inhibitor); Aprotinin (serine protease inhibitor); Bestatin (aminopeptidase inhibitor); Leupeptin (serine and cysteine protease inhibitor); E-64 (selective cysteine protease inhibitor, such as cathepsin B and L); and protease inhibitor cocktail (AEBSF; serine proteases inhibitor; Phenanthroline, metalloproteases inhibitor; Pepstatin A, acid proteases inhibitor, Leupeptine, Bestatin and E-64) from Sigma Aldrich code P9599 (St Louis, MO, United States).

Gandullo J., Monreal J.A., Álvarez R., Díaz I., García-Mauriño S, & Echevarría C. (2019). Anionic Phospholipids Induce Conformational Changes in Phosphoenolpyruvate Carboxylase to Increase Sensitivity to Cathepsin Proteases. Frontiers in Plant Science, 10, 582.

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Inhibiting Proteolysis in Plant Cells

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CHX was used to inhibit protein synthesis in plant cells. One millimolar cysteine protease inhibitor E-64 ((1S,2S)-2-(((S)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl) cyclopropane carboxylic acid; Product Code E3132), 1 mM serine protease inhibitor AEBSF (4-(2-Aminoethyl) benzene-sulfonyl fluoride hydrochloride; Product Code A8456), 0.5 mM metallo-aminopeptidase inhibitor Bestatin (Product Code B8385), 1 mM acid protease inhibitor Pepstatin A (Product Code P4265), 1 mM metalloprotease inhibitor 1,10-phenanthroline monohydrate (Product Code P9375) and Plant Protease Inhibitor Mixture (Product code P9599; PI; contains AEBSF, Bestatin, E-64, Leupeptin, Pepstatin A, 1,10-Phenanthroline) were purchased from Sigma-Aldrich and used to inhibit proteolysis.

Xia Y., Ma Z., Qiu M., Guo B., Zhang Q., Jiang H., Zhang B., Lin Y., Xuan M., Sun L., Shu H., Xiao J., Ye W., Wang Y., Wang Y., Dong S., Tyler B.M, & Wang Y. (2020). N-glycosylation shields Phytophthora sojae apoplastic effector PsXEG1 from a specific host aspartic protease. Proceedings of the National Academy of Sciences of the United States of America, 117(44), 27685-27693.

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Protease Inhibitor Cocktail for In Vitro and In Vivo Studies

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4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlorine (AEBSF, A8456), aprotinin (A1153), bestatin (Sigma B8385), pepstatin A (P5318), N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64, E3132), leupeptin (L2884), N-ethylmaleimide (NEM, E3876), calpeptin (C8999), calpain inhibitor-I (A6185), -II (A6060), -III (M6690), pan-caspase inhibitor (V116), BAPTA-AM (A1076), ionomycin (I0634) were supplied by Sigma. Calpistatin peptide (208902), negative control peptide (208904), elastase inhibitor IV (324759) and cathepsin G inhibitor I (219372) were supplied by Calbiochem. Tryptase inhibitor nafamostat mesylate (NM, 3081) was supplied by Tocris. AEBSF, aprotinin, bestatin, pepstatin A, E64, leupeptin and NEM were used in vitro at concentrations according to manufacturer’s instructions (Sigma Inhib1). calpeptin, calpain, NE, CG, caspase and tryptase inhibitors, BAPTA-AM were used at 100 μM during in vitro studies. Calpistatin and control peptide were used at 10 μM during in vitro studies. Doses of inhibitors used in vivo and details of all other reagents used are described in other method sections.

Scott I.C., Majithiya J.B., Sanden C., Thornton P., Sanders P.N., Moore T., Guscott M., Corkill D.J., Erjefält J.S, & Cohen E.S. (2018). Interleukin-33 is activated by allergen- and necrosis-associated proteolytic activities to regulate its alarmin activity during epithelial damage. Scientific Reports, 8, 3363.

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Mitochondrial Respiration Complex Assay

Cited 1 time

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Cells were rinsed twice in ice-cold PBS, lysed with 0.5 mL buffer A (50 mmol/L Tris, 100 mmol/L KCl, 5 mmol/L MgCl₂, 1.8 mmol/L ATP, 1 mmol/L EDTA, pH 7.2) containing the protease inhibitor cocktail III [100 mmol/L AEBSF, 80 mmol/L aprotinin, 5 mmol/L bestatin, 1.5 mmol/L E-64, 2 mmol/L leupeptin and 1 mmol/L pepstatin (MerckMillipore, Milan, Italy) 1 mmol/L PMSF, 250 mmol/L NaF]. Samples were centrifuged at 650× g for 3 min at 4 °C, supernatants were transferred into a new tube series and centrifuged at 13,000× g for 5 min at 4 °C. The supernatants were discarded, the pellets containing mitochondria, after a washing step with 0.5 mL buffer A, were re-suspended in 0.25 mL buffer B (250 mmol/L sucrose, 15 μmol/L K₂HPO₄, 2 mmol/L MgCl₂, 0.5 mmol/L EDTA, 5% w/v BSA). 50 μL were sonicated and used for protein quantification. The activity of mitochondria respiration complexes was evaluated according to [18 (link)]. Results were expressed as nmol red cit c/min/mg mitochondrial proteins.

Roux C., Mucciolo G., Kopecka J., Novelli F., Riganti C, & Cappello P. (2021). IL17A Depletion Affects the Metabolism of Macrophages Treated with Gemcitabine. Antioxidants, 10(3), 422.

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Isolation and Respiration Assay of Mitochondria

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According to Wibom et al. [20 (link)], cells were washed twice in ice-cold 0.1 M phosphate-buffered saline (PBS), then lysed in 0.5 mL buffer A (50 mmol/L Tris, 100 mmol/L KCl, 5 mmol/L MgCl₂, 1.8 mmol/L ATP, 1 mmol/L EDTA, pH 7.2), supplemented with protease inhibitor cocktail III [100 mmol/L AEBSF, 80 mmol/L aprotinin, 5 mmol/L bestatin, 1.5 mmol/L E-64, 2 mmol/L leupeptin and 1 mmol/ L pepstatin (Merck KGaA, Darmstadt, DE)], 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 250 mmol/L NaF. Samples were clarified by centrifuging at 650× g for 3 min at 4 °C, and the supernatant was collected and centrifuged at 13,000× g for 5 min at 4 °C. The new supernatant was discarded, the pellet containing mitochondria was washed in 0.5 mL buffer A and re-suspended in 0.25 mL buffer B (250 mmol/L sucrose, 15 mmol/L K₂HPO₄, 2 mmol/L MgCl₂, 0.5 mmol/L EDTA, 5% w/v bovine serum albumin). Afterwards, the activity of mitochondria respiration complexes was measured according to Wibom et al. [20 (link)].

Allocco A.L., Bertino F., Petrillo S., Chiabrando D., Riganti C., Bardelli A., Altruda F., Fiorito V, & Tolosano E. (2022). Inhibition of Heme Export and/or Heme Synthesis Potentiates Metformin Anti-Proliferative Effect on Cancer Cell Lines. Cancers, 14(5), 1230.

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Mitochondrial Enzyme Activity Assay

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According to [22 (link)], cells were washed twice in ice-cold 0.1 M phosphate-buffered saline (PBS), then lysed in 0.5 mL buffer A (50 mmol/L Tris, 100 mmol/L KCl, 5 mmol/L MgCl₂, 1.8 mmol/L ATP, 1 mmol/L EDTA, pH 7.2), supplemented with protease-inhibitor cocktail III [100 mmol/L AEBSF, 80 mmol/L aprotinin, 5 mmol/L bestatin, 1.5 mmol/L E-64, 2 mmol/L leupeptin and 1 mmol/L pepstatin (Merck, Darmstadt, Germany), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 250 mmol/L NaF. Samples were clarified by centrifuging at 650× g for 3 min at 4 °C, and the supernatant was collected and centrifuged at 13,000× g for 5 min at 4 °C. The new supernatant was discarded and the pellet containing mitochondria was washed in 0.5 mL buffer A and re-suspended in 0.25 mL buffer B (250 mmol/L sucrose, 15 μmol/L K₂HPO₄, 2 mmol/L MgCl₂, 0.5 mmol/L EDTA, 5% w/v bovine serum albumin). A 100 μL aliquot was sonicated and used for the measurement of protein content. The remaining not-sonicated part was used to measure the electron transport chain (ETC) complexes I–IV activities according to [22 (link)]. Results were expressed as nmol NAD+/min/mg mitochondrial protein for complex I, nmol cyt c reduced/min/mg mitochondrial protein for complexes II–III, and nmoles cyt c oxidized/min/mg mitochondrial protein for complex IV.

Petrillo S., De Giorgio F., Kopecka J., Genova T., Fiorito V., Allocco A.L., Bertino F., Chiabrando D., Mussano F., Altruda F., Munaron L., Riganti C, & Tolosano E. (2021). Endothelial Heme Dynamics Drive Cancer Cell Metabolism by Shaping the Tumor Microenvironment. Biomedicines, 9(11), 1557.

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Western blot analysis of autophagy markers

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Commercially available antibodies were used in Western blots (WB), for the detection of sacsin (Anti-sacsin AbC ab181190. Abcam, Cambridge, UK), spectrin (Anti-spectrin MAB1622 Merck Millipore, Burlington, MA, USA), ubiquitinated proteins (Anti-ubiquitin ab134953, Abcam), p62 (Anti-p62/SQSTM1 P0067 Merck Millipore), LC3I-II (Anti-LC3A/B ab58610. Abcam), HA (anti-HA Epitope Tag 16b12, Biolegend, San Diego, CA, USA). Secondary antibodies included HRP-conjugated anti-mouse and anti-rabbit IgG (Amersham Bioscience, Buckinghamshire, UK).
In vitro treatments were carried out with different compounds: 1 µM MG-132 24 hours or 3 hours (Merck Millipore), 20 µM Chloroquine (CQ) 24 hours (Merck Millipore) or together (0.25 µM MG-132 + 10 µM CQ, 18 hours); protease inhibitors (5 µM E64, 10 µM Bestatin, 5 µM Pepstatin, 48 hours (Merck Millipore)). After the treatments, cells were harvested and lysed for biochemical assays.

Longo F., Ritis D.D., Miluzio A., Fraticelli D., Baets J., Scarlato M., Santorelli F.M., Biffo S., & Maltecca F. (2021). Sacsin cotranslational degradation causes autosomal recessive spastic ataxia of Charlevoix-Saguenay.

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Protease Inhibitors and Substrates

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Phenylmethylsulfonyl fluoride (PMSF), leupeptin, Bestatin, E-64c, aprotinin, chymostatin and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma (St. Louis, MO). Protease substrates, t-butyloxycarbonyl (Boc)-L-Phe-Ser-Arg 4-methylcoumaryl-7 -amide (MCA), Leu-MCA, benzyloxycarbonyl (Z) -L-Phe -Arg-MCA and (7-methoxycoumarin-4-yl) acetyl (MOCAc)-L-Arg-Pro-Lys-Pro-Tyr -Ala-norvalyl (Nva) -Trp-Arg-N ε -(2,4 -dinitrophenyl) -L-Lys (Lys(Dnp)) -NH2 were purchased from Peptide Institute (Osaka, Japan).

Kamemura N., Takashima M., Morita H., Matsumoto K., Saito H, & Kido H. (2015). Measurement of allergen-specific secretory IgA in stool of neonates, infants and toddlers by protection against degradation of immunoglobulins and allergens. The journal of medical investigation : JMI, 62(3-4).

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MAPK Protein Profiling in PMNs

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The Proteome Profiler™ human phospho-MAPK slide array kits (R&D Systems Inc., Minneapolis, MN) were used for detection of 24 different MAPK proteins in PMNs. Whole-cell lysates were prepared according to the manufacturer’s instructions. Briefly, neutrophils were left untreated or were infected with LVS, and 2, 10 or 24 hours later neutrophils were pelleted by centrifugation at 250 g. Cell pellets were resuspended in lysis buffer containing aprotinin, leupeptin, PMSF, AEBSF, levamisole, bestatin, E-64, and pepstatin A (Sigma-Aldrich, St. Louis, MO), supplemented with the Pierce Halt™ phosphatase inhibitor cocktail (sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate) (Thermo Fisher Scientific, Waltham, MA). Protein concentrations were determined by performing a Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. Array dot blots were processed according to the manufacturer’s protocol.

Kinkead L.C., Krysa S.J, & Allen L.A. (2022). Neutrophil Survival Signaling During Francisella tularensis Infection. Frontiers in Cellular and Infection Microbiology, 12, 889290.

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Isolation and Lysis of Podosomal Structures

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Primary OCLs were cultured in six-well plates and in some samples infected with adenoviruses as described. Cells were lysed in 200 μl/well lysis buffer (20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton, and 1 mM sodium vanadate with protease inhibitors [1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 40 μM bestatin, 15 μM E-64, 20 μM leupeptin, 15 μM pepstatin; Sigma-Aldrich]) on ice for 10 min with gentle shaking. Cell bodies and cytoplasm were removed and saved. The remaining adherent podosomal structures were washed gently three times with 200 μl of lysis buffer. Lysis buffer was completely removed, and the remaining cell structures were solubilized in 100 μl/well of 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1% SDS, and 1% sodium deoxycholate with protease inhibitors.

Finkelshtein E., Lotinun S., Levy-Apter E., Arman E., den Hertog J., Baron R, & Elson A. (2014). Protein tyrosine phosphatases ε and α perform nonredundant roles in osteoclasts. Molecular Biology of the Cell, 25(11), 1808-1818.

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