Ketoprofen resolution by enzymatic estirification and hydrolysis of the ester product

2006 ◽  
Vol 11 (3) ◽  
pp. 211-214 ◽  
Author(s):  
Jin Chuan Wu ◽  
Hou Ran Low ◽  
Yujun Leng ◽  
Yvonne Chow ◽  
Ruijiang Li ◽  
...  
Keyword(s):  
Hydrolysis Of ◽  
Ester Product

Assay of the Esterase Activity of Thrombin, Plasmin and Trypsin with a Chromogenic Substrate p-Nitrobenzyl p-Toluenesulfonyl-L-Arginine

1977 ◽  
Vol 37 (02) ◽  
pp. 253-261 ◽  
Author(s):  
Tadashi Aogaichi ◽  
Gerhard W. E Plaut
Keyword(s):  
Product Formation ◽  
Enzyme Concentration ◽  
Bovine Thrombin ◽  
Absorbance Change ◽  
Bovine Trypsin ◽  
Nitrobenzyl Alcohol ◽  
Human Thrombin ◽  
Hydrolysis Of ◽  
Ester Product ◽  
Esterase Activities

Summaryp-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 μmol/ml of the ester is equivalent to an absorbance change of 4.45 cm–1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30°, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 μg/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30°, Kmis 14 μM and Vmax is 0.037 μmol/min/NIH unit/ml for bovine thrombin, Km is 78 μM and Vmax is 0.31 μmol/min/CTA unit/ml for human plasmin, and Km is 12 μM and Vmax is 138 μmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 μmol p-nitrobenzyl alcohol formed/10 min/NIH unit.

The partitioning of tetrahedral intermediates of the hydrolysis of benzimidatonium ions in acid solutions

1980 ◽  
Vol 58 (22) ◽  
pp. 2318-2325 ◽  
Author(s):  
Robert A. McClelland ◽  
Janet P. Potter
Keyword(s):  
Strong Acid ◽  
Major Product ◽  
Dilute Acid ◽  
Acid Product ◽  
Aromatic Substituent ◽  
Tetrahedral Intermediate ◽  
Zwitterionic Form ◽  
Hydrolysis Of ◽  
Ester Product ◽  
Amide Acetals

The products (a benzoate ester or an N,N-dimethylbenzamide) have been accurately determined for the hydrolysis of the O-ethyl-N,N-dimethylbenzimidatonium ion (1) and its 4-nitro derivative (2) in dilute and moderately concentrated H2SO4 solutions. In dilute acid the ratio of amide:ester is very small (0.0005–0.001). It is proposed that the ester product arises from the zwitterionic form of the tetrahedral intermediate of the hydrolysis. The small amount of amide is due to an SN2 hydrolysis reaction and to the non-catalyzed breakdown of the neutral form of the tetrahedral intermediate, the relative contributions of the two being unknown. Although ester remains the major product throughout, at some intermediate acid concentration (20% H2SO4 for 1 and 35% H2SO4 for 2) a sharp increase in the amide:ester ratio is observed, followed by a levelling-off. This change is shown to be associated with tetrahedral intermediate partitioning, and not the SN2 reaction. It is argued that the strong acid product ratio represents the breakdown of the tetrahedral intermediate via cationic transition States. Two kinetically equivalent reactions are involved, the breakdown of the N-protonated tetrahedral intermediate producing a protonated ester and amine and the H+-catalyzed breakdown of the neutral tetrahedral intermediate producing a protonated amide and alcohol. Analogous pathways are also observed for the decomposition in acid of the amide acetals ArC(OMe)2NMe2, and the two systems are compared. A similar effect of the aromatic substituent on the partitioning ratio is noted, but the tetrahedral intermediate exhibits a greater tendency for amine expulsion. A kinetic analysis produces the estimate of 1011 s−1 for the rate constant for the breakdown of the zwitterionic form of the tetrahedral intermediate. This very large rate explains why its decomposition remains important even in concentrated acids, where the amount of tetrahedral intermediate which actually exists as zwitterion must be extremely low.

Fine Structural Localization of Acyltransferases Involved in Lipid Synthesis in Intestinal Mucosa.

1970 ◽  
Vol 28 ◽  
pp. 54-55
Author(s):  
R. J. Barrnett ◽  
J. A. Higgins
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Lipid Synthesis ◽  
Mucosal Cell ◽  
Structural Localization ◽  
Morphological Studies ◽  
Mucosal Cells ◽  
Initial Appearance ◽  
Sh Group ◽  
Hydrolysis Of ◽  
Intestinal Mucosal Cells

The main products of intestinal hydrolysis of dietary triglycerides are free fatty acids and monoglycerides. These form micelles from which the lipids are absorbed across the mucosal cell brush border. Biochemical studies have indicated that intestinal mucosal cells possess a triglyceride synthesising system, which uses monoglyceride directly as an acylacceptor as well as the system found in other tissues in which alphaglycerophosphate is the acylacceptor. The former pathway is used preferentially for the resynthesis of triglyceride from absorbed lipid, while the latter is used mainly for phospholipid synthesis. Both lipids are incorporated into chylomicrons. Morphological studies have shown that during fat absorption there is an initial appearance of fat droplets within the cisternae of the smooth endoplasmic reticulum and that these subsequently accumulate in the golgi elements from which they are released at the lateral borders of the cell as chylomicrons.We have recently developed several methods for the fine structural localization of acyltransferases dependent on the precipitation, in an electron dense form, of CoA released during the transfer of the acyl group to an acceptor, and have now applied these methods to a study of the fine structural localization of the enzymes involved in chylomicron lipid biosynthesis. These methods are based on the reduction of ferricyanide ions by the free SH group of CoA.

Lattice imaging and pore structure of β ferric oxyhydroxide

1978 ◽  
Vol 36 (1) ◽  
pp. 264-265
Author(s):  
T. Baird ◽  
J.R. Fryer ◽  
S.T. Galbraith
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Bulk Material ◽  
Model Structure ◽  
Bulk Crystals ◽  
Lattice Imaging ◽  
Thin Sections ◽  
Ferric Oxyhydroxide ◽  
Resolution Electron ◽  
Hydrolysis Of

Introduction Previously we had suggested (l) that the striations observed in the pod shaped crystals of β FeOOH were an artefact of imaging in the electron microscope. Contrary to this adsorption measurements on bulk material had indicated the presence of some porosity and Gallagher (2) had proposed a model structure - based on the hollandite structure - showing the hollandite rods forming the sides of 30Å pores running the length of the crystal. Low resolution electron microscopy by Watson (3) on sectioned crystals embedded in methylmethacrylate had tended to support the existence of such pores.We have applied modern high resolution techniques to the bulk crystals and thin sections of them without confirming these earlier postulatesExperimental β FeOOH was prepared by room temperature hydrolysis of 0.01M solutions of FeCl3.6H2O, The precipitate was washed, dried in air, and embedded in Scandiplast resin. The sections were out on an LKB III Ultramicrotome to a thickness of about 500Å.

Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors

2019 ◽  
Vol 47 (6) ◽  
pp. 1733-1747 ◽  
Author(s):  
Christina Klausen ◽  
Fabian Kaiser ◽  
Birthe Stüven ◽  
Jan N. Hansen ◽  
Dagmar Wachten
Keyword(s):  
Cyclic Amp ◽  
Adenosine Monophosphate ◽  
Adenylyl Cyclases ◽  
Temporal Precision ◽  
Fluorescent Biosensors ◽  
Subcellular Domains ◽  
Spatio Temporal ◽  
Hydrolysis Of ◽  
Shed Light ◽  
Cyclic Amp Signaling

The second messenger 3′,5′-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.

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