Phenylmethylsulfonyl fluoride

Resuspend the resin in 50 /A of elution buffer E (20 mM Tris-HCl [pH 7.5], 100 mM KC1, 5 mM MgCl2, 0.5 mM /J-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 250 mM imidazole, 10% (v/v) glycerol, 1X Complete Protease Inhibitor Mix tablets [Roche]), briefly vortex, and elute the bound proteins by mixing on a rotator at 4° for 20 min. Please note that a concentration of imidazole and the kind of Roche tablets differs from BB buffer. 

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

SERINE DEHYDRATASE SERINE DEHYDRATASE SERINE DEHYDROGENASE Serine esterase inhibitor, irreversible, PHENYLMETHYLSULFONYL FLUORIDE SERINE HYDROXYMETHYLTRANSFERASE SERINE PALMITOYLTRANSFERASE Serine protease inhibitor, irreversible, PHENYLMETHYLSULFONYL FLUORIDE Serine proteinase inhibitor,... 

Three preliminary experiments were carried out the first done as described above, the second utilized a modified buffer containing protectants, and the third utilized both the modified buffer and a hemoglobin gel step to remove proteases. The modified buffer had the following recipe 50 mM EPPS-KOH buffer at pH 8.6, 20% (v/v) glycerol, 1.0 mM EDTA, 1.0 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM leupeptin, and 0.05% / -mercaptoethanol. 

Selective chemical change of the serine—OH group to cysteine—SH in enzymes can be performed with extremely reactive serine residues in the active sites by the use of phenylmethylsulfonyl fluoride and, subsequently, thioacetic acid (Polgar and Bender, 1966). This selective chemical modification demonstrates the essential role of an—OH... 

Cut the tissue into small pieces and add these to 50 ml 1 mmol/1 NaHC03 (pH 8.2) Add phenylmethylsulfonyl fluoride (PMSF) to a final PMSF concentration of 1 mmol/1 Stir 15 min at 4 °C... 

Elastase Bowman-Birk inhibitor, chicken ovoinhibitor, diisopropyl fluorophosphates, elastatinal, phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor... 

Phenylmethylsulfonyl fluoride (PMSF) stock (100X) add 174 mg PMSF to 10 mL 100% ethanol. Store at 4°C. 

The original DEBS 1-TE cell-free system allowed several other features of polyketide chain extension to be examined by using well-known inhibitors of specific enzyme activities. Incubation of DEBS 1 -TE with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) resulted in a significant decrease in biosynthetic activity. This result emphasized the mechanistic similarity between the targeted TE domain and the serine protease enzymes. Addition of the fatty acid inhibitor cerulenin [38] also reduced production of the lactone, consistent with the evolutionary kinship between these two classes of enzymes. Inhibition of DEBS 1+TE by cerulenin was also reported [33],... 

Phenylmethylsulfonyl fluoride (PMSF) and yeast AO were purchased from Sigma (St. Louis, MO), and the alcohol substrates were from J.T. Baker (Phillipsburg, NJ). All other reagents were of the highest purity commercially available. Protein was measured by the method of Lowry (7) using bovine serum albumin as standard. 

Experimental evidence that protein structures are involved in stratum corneum cell cohesion was presented by Bisset et al.9 They induced cell dissociation in pig and human nonpalmo-plantar stratum corneum by means of incubation of the tissue in the presence of the zwitterionic surfactant 6-octadecyldimethyl ammoniohexanoate. Cell dissociation could not be induced when the tissue had been pretreated with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The fact that cell dissociation was found only in the presence of EDTA suggested a role also for calcium in stratum corneum cell cohesion. 

Representatives of this class of inhibitors are DFP (diisopropylfluoro-phosphate), PMSF (phenylmethylsulfonyl fluoride), APMSF (4-aminophe-nyl)-methanesulfonyl, AEBSF (4-(2-aminoethyl)-benzenesulfonyl fluoride), FK-448 (4-(4-isopropylpiperadinocarbonyl) phenyl 1,2,3,4,-tetrahydro-1-naphthoate methanesulfonate), camostat mesilate (7V,/V -dimethyl carba-moylmethyl-/)-(/> -guanidino-benzoyloxy)phenylacetate methanesulfonate), and Na-glycocholate. The organophosphorus inhibitors DFP and PMSF are potent irreversible inhibitors of serine proteases. Due to their... 

The assay was conducted by adding 10 / L of serum or plasma to 100 tiL containing 500 fiM substrate in 100 mM Mops-NaOH (pH 7.0), 1 mM phenylmethylsulfonyl fluoride, and 3 mM o-phenanthroline. The reaction mixture was incubated at 37°C for an hour, and then 60 fih was directly injected into the column. 

The reaction mixture contained 1 mg of microsomal protein per milliliter, 50 mM Tris-HCl buffer (pH 7.4), 10 mM MgCl2,10 mM 1,4-saccharolactone, 1 mM phenylmethylsulfonyl fluoride, 0.04% Triton X-100, 10 mM UDP-glucuronic acid, and 0.1 to 0.4 mM substrate in a final volume of 250 pL. The reaction was initiated by the addition of UDP-glucuronic acid. After 20... 

Carboxylamidase activity toward p-nitroacetanilide has been detected in different insect species from the orders Lepidoptera, Orthoptera, and Dictyoptera. The carboxylamidase from fall army worm larvae has been purified. The purified enzyme is a monomer with a molecular weight of 59,000-60,000 Da. The enzyme is inhibited by the hydrolase inhibitors paraoxon, triphenyl phosphate, eserine, and phenylmethylsulfonyl fluoride, showing I50 values of 4.7 iM, 0.2 mM, 16 iM, and 90 iM, respectively. Activity is also completely inhibited by the organophosphorus insecticides profenfos and dichlorvos at 0.1 mM. The enzyme is active toward other amides, such as acetanilide and phenacetin, and various a- and p-naphtholic esters. Based on the purification factor, substrate specificity, and sensitivity to hydrolase inhibitors, the carboxylamidase appears to be different from carboxy-lesterases in the fall army worm (Yu and Nguyen, 1998). 

Protein extraction methods are critical. In previous studies various extraction procedures have been tested. Paris et al. [20] could show that after breaking A. alternata cells, the use of carbonate buffer supplemented with protease inhibitors (phenylmethylsulfonyl fluoride) and phenol-binding components... 

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