Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple and specific method for the estimation of trypsin in human duodenal juice was described. The procedures are as follows: add 10 ul of undiluted sample, measure 2.0 ml of substrate solution of benzoylarginine p-nitroanilide (BAPNA) 0.5 mg/ml in a Tris buffer, incubate at 37 degrees C for 10 minutes, then terminate tryptic activity with 2.0 ml of 30% v/v acetic acid, and read absorbance at 410 nm by a spectrophotometer. Coexistence of bile pigments, chymotrypsin or elastase did not interfere the estimation of tryptic activity in duodenal juice. Reproducibility (both within- and between-assay variances less than 8%), recovery (mean of 100%) and stability of the enzyme activity after 3 weeks at -20 degrees C with glycerol (96% of the initial activity) were sufficient for clinical use. The amidase activity of trypsin estimated with BAPNA as substrate correlated well both to the esterase activity measured with p-toluenesulfonyl-L-arginine methyl ester (TAME) as substrate and to the immunoreactivity determined by radioimmunoassay in human duodenal juice. Good correlation between total outputs of amylase and trypsin were observed in 29 patients undergoing pancreozymin secretin test. The present assay technique will provide simple and reliable means of measuring trypsin in duodenal fluid and of mutual checks of the secretory capacity of pancreatic enzymes and will increase diagnostic accuracy of pancreozymin secretin test.
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PMID:A simple and specific determination of trypsin in human duodenal juice. 615 4

A canine model of bile-induced pancreatitis has been employed to investigate time-dependent changes in the molecular forms of trypsin in blood and ascitic fluid in this disease. The distribution of immunoreactive trypsin as trypsinogen and trypsin bound to plasma inhibitors in ascitic fluid and plasma during the course of the disease has been investigated by means of a radioimmunoassay for canine pancreatic cationic trypsin. In addition, trypsinlike amidase activity was determined in plasma and ascitic fluid using Z-Gly-Gly-Arg-beta-Nap as substrate. Early plasma and ascitic fluid samples in four dogs that died contained primarily trypsinogen, while extensive activation of trypsinogen to alpha 2-macroglobulin and alpha 1-protease inhibitor-bound trypsin occurred in the course of the disease. A fifth dog survived and showed little activation of trypsinogen. In the four dogs that died, the levels of trypsinlike amidase activity in the ascitic fluid were substantial throughout the course of the disease. The plasma levels of trypsinlike activity in these animals were much lower, but increased during the disease process. The dog that survived had lower concentrations of trypsinlike activity in ascitic fluid and plasma. These results suggest that activation of trypsinogen resulting in inhibitor-bound forms of trypsin in ascitic fluid and plasma is important in the pathogenesis of acute pancreatitis.
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PMID:Immunoreactive forms of cationic trypsin in plasma and ascitic fluid of dogs in experimental pancreatitis. 617 Feb 31

It was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.31), plasmin (E C 3.4.21.7) and trypsin (E C 3.4.21.4) depended on the substrate used. No effect of heparin on the amidase activity of urokinase and trypsin was observed when Pyro Glu-Gly-Arg-p-nitroanilide (S-2444) and alpha-N-acetyl-L-lysine-p-nitroanilide (ALNA) were used as substrates. Heparin acted as a uncompetitive inhibitor of trypsin (Ki = 1.2 X 10(-6) M), plasmin (Ki = 4.9 X 10(-6) M) and urokinase (Ki = 1.0 X 10(-7) M) when Bz-Phe-Val-Arg-p-nitroanilide (S-2160), H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and plasminogen, respectively, were used as substrates. These results, as well as the data obtained by studying the effect of the simultaneous presence of heparin and competitive inhibitors suggest that although heparin is not bound at the active center of these enzymes, it may influence the effectivity of catalysis.
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PMID:Kinetic study of the effect of heparin on the amidase activity of trypsin, plasmin and urokinase. 622 10

Rheumatoid synovial fluid contains an activator of latent collagenase from culture medium of pig synovium. The activator was purified by gel chromatography on Ultrogel AcA 44 and affinity chromatography on soybean trypsin inhibitor coupled to Sepharose 4B. The purified material was homogeneous on SDS-polyacrylamide gel electrophoresis with Mr 88 000. The activator had limited proteolytic activity against azo-casein, but showed amidase activity on Pro-Phe-Arg-NMec, Z-Phe-Arg-NMec, D-Val-Leu-Arg-NPhNO2 and D-Pro-Phe-Arg-NPhNO2, with an optimum at pH 8.0. Activity was completely inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, leupeptin and Pro-Phe-Arg-CH2Cl, whereas lima bean trypsin inhibitor, Tos-Lys-CH2Cl, a specific inhibitor of factor XIIa from maize, EDTA and iodoacetate were not inhibitory. These properties of the activator suggested that it might be plasma kallikrein (EC 3.4.21.34), and the possibility was further examined. The activator was treated with [3H]diisopropyl fluorophosphate, and run in SDS-polyacrylamide gel electrophoresis with reduction; a radioautograph of the gel showed a pair of [3H]diisopropyl phosphoryl-labelled bands (Mr 36 000 and 34 000) identical to those obtained with authentic plasma kallikrein. Double immunodiffusion with monospecific antiserum against human plasma kallikrein confirmed the identification. This is the first demonstration of collagenase-activating activity of plasma kallikrein, and raises the possibility that activation of prokallikrein in the inflamed joint space may contribute to the disease process not only by the production of bradykinin, but also by activating latent collagenase.
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PMID:Identification of plasma kallikrein as an activator of latent collagenase in rheumatoid synovial fluid. 627 61

A new reliable human blood treatment was established for quantitative endotoxin assay using synthetic chromogenic substrate [Boc-Leu-Gly-Arg-rho-nitroanilide]. Addition of perchloric acid in a final concentration of 1.25% to platelet-rich plasma or serum in a 2:1 volume ratio completely eliminated nonspecific amidase activities as well as inhibitors. By this method, the recovery of added endotoxin was nearly 100%, and was almost independent of sample dilutions and anticoagulants.
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PMID:Perchloric acid treatment of human blood for quantitative endotoxin assay using synthetic chromogenic substrate for horseshoe crab clotting enzyme. 628 88

A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed.
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PMID:Rhodamine-based compounds as fluorogenic substrates for serine proteinases. 634 11

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.
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PMID:A functionally active heavy chain derived from human high molecular weight urokinase. 634 38

Three acidic arginine esterases have been isolated from the venom of the Western Diamondback rattlesnake (Crotalus atrox). These components demonstrated marked differences in ionic characteristics, as noted by KCl gradient elution from a DEAE-Sephadex A-50 column. Molecular weights, as determined from gel permeation chromatography, were estimated at 25,100 (fraction D), 24,000 (B); and 22,900 (F) for the three separate enzymes. The two larger enzymes (B and D) exhibited similar activities toward the synthetic substrates alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-DL-arginine-p-nitroanilide. Hydrolysis rates were similar to commercial trypsin preparations. Fraction F exhibited a markedly lower activity as an arginine esterase and negligible activity as an arginine amidase. Arginine esterase activity was evident for all three enzymes in the presence of ethylenediamine tetra-acetic acid.
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PMID:Purification of three arginine esterases from the venom of the Western Diamondback rattlesnake (Crotalus atrox). 681 57

Dimethyl sulfoxide produces an opposite effect on the esterase and amidase activities of bovine thrombin. The esterase activity is increased by two fold but the amidase activity is decreased to 9% of the initial activity in 20% dimethyl sulfoxide. The stimulation of the esterase activity is due to the change in Vmax rather than Km for the substrate p-Tosyl-L-Arginine methyl ester. The inhibition of the esterase activity of thrombin by NaCl is not affected due to the addition of dimethyl sulfoxide. Ki for NaCl, 0.03 M, is the same for both in the absence and in the presence of 10% dimethyl sulfoxide. The catalytic activity of thrombin is inhibited by heparin. This effect is significantly decreased by dimethyl sulfoxide. The dissociation constant of heparin-thrombin complex, measured in the absence and in the presence of 10% dimethyl sulfoxide are 4 nM and 28 nM respectively. Thermal stability of thrombin, determined by monitoring catalytic activity, is increased in the presence of dimethyl sulfoxide. The enhancement of the fluorescence intensity of thrombin in the presence of dimethyl sulfoxide reflects the contribution of more exposed tryptophanyl residues. The alteration of the conformation of the enzyme structure due to the perturbation of the aqueous medium by dimethyl sulfoxide, has been attributed to these observed effects.
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PMID:The catalytic activity and physical properties of bovine thrombin in the presence of dimethyl sulfoxide. 684 75

We studied the effect of ions on the ability of purified human urinary kallikrein to cleave its natural substrate (kininogen) as well as two synthetic substrates, tosylarginine [3H]methyl ester and Pro-Phe-Arg-[3H]benzylamide. The kininogenase activity of kallikrein is markedly dependent upon the concentration of cations in vitro. Kininogenase activity is very low when measured in a low electrolyte buffer. The addition of cations to the reaction mixture increases activity by up to 27-fold. Maximum activity is achieved with 100 mM sodium, 100 mM potassium, or 20 mM magnesium. The activity is stable at higher concentrations of cation. Renal kallikrein is believed to act within distal tubular fluid in vivo. The concentration of cations in this fluid varies widely in response to alterations in salt and water metabolism. Thus, the relationship of kininogenase activity to the concentration of cations demonstrated in vitro may be relevant to the activity of kallikrein at its presumed site of action in the kidney. In separate experiments, we evaluated the effect of ions on the amidase and esterase activities of kallikrein which are the basis of several assays in routine use for physiological studies. In contrast to their stimulatory effect on kininogenase activity, cations inhibit amidase and to a lesser extent esterase activity. Additional studies indicate that urinary cations probably account entirely for the well known ability of normal urine to inhibit the amidase and esterase activities of kallikrein.
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PMID:The effect of cations on the activity of human urinary kallikrein. 692 Dec 2


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