Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The monoclonal antibody (mAb) 2G8 (subclass IgG2a) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys flesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1.8) from human serum. Binding of mAb 2G8 to the globular G2 form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.
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PMID:The monoclonal antibody 2G8 is carbohydrate-specific and distinguishes between different forms of vertebrate cholinesterases. 204 Feb 91

1. The influences of enzyme treatments (trypsin and collagenase) on responses to perfused acetylcholine were examined on physically isolated single Aplysia neurons, using the voltage-clamp, internal perfusion, and rapid external perfusion technique. 2. During treatment with trypsin (0.025 to 0.1%) for 10 to 30 min at room temperature (22 to 25 degrees C), the peak amplitude of the Na current induced by acetylcholine increased in a time- and dose-dependent manner, and the decay in the continued presence of acetylcholine was slowed. This effect of trypsin treatment was irreversible after washing for 60 min without enzyme. 3. Edrophonium, a cholinesterase inhibitor, has previously been shown to augment the Na acetylcholine response in this preparation by inhibition of acetylcholinesterase. After treatment of the neuron with trypsin, the augmentation after edrophonium was abolished. Furthermore, in the presence of edrophonium, trypsin also failed to increase the response. The dose-response curve for acetylcholine after treatment of trypsin was similar to that in the presence of edrophonium. These results suggest that the modification of the current response by trypsin is a result of removal of cholinesterase activity from the membrane. 4. In contrast to the effects of trypsin, collagenase (0.03 to 0.1%) for 10 to 60 min did not change the current amplitude of the acetylcholine response. However, collagenase treatment did alter the kinetics of the acetylcholine response in a dose-dependent manner, in that the rate of decay was accelerated. A similar acceleration was seen in the acetylcholine responses on other neurons which were due to Cl or K currents, suggesting that the effect was independent on the type of channel. This effect of collagenase was reversible after 30 to 60 min of washing of the neuron. 5. In the presence of edrophonium or after the treatment with trypsin, collagenase still accelerated the current kinetics of the acetylcholine response, indicating that cholinesterase activity is not related to this effect. Furthermore, heated collagenase (presumably inactivated) had a similar action, suggesting that the enzymatic activity of collagenase is not related to the modification of the response. 6. These results suggest that Aplysia acetylcholinesterase is sensitive to trypsin but not to collagenase. However, the preparation of a collagenase used in these studies contains some factor which alters the response to acetylcholine, but this effect is reversible and unrelated to enzymatic activity.
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PMID:Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons of Aplysia californica. 216 51

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.
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PMID:Mutagenesis of essential functional residues in acetylcholinesterase. 221 85

Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.
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PMID:Amphiphilic and hydrophilic molecular forms of acetylcholinesterase in membranes derived from sarcoplasmic reticulum of skeletal muscle. 237 90

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.
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PMID:O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues. 250 78

Unilamellar liposomes prepared from sn-3-(dimyristoyl)phosphatidylcholine (DMPC) in the presence and absence of acetylcholinesterase were examined by ESR for lipid/protein interactions. Using 5-, 12-, and 16-doxyl stearic acid probes incorporated into the phospholipid bilayers, no measurable differences in the gel to liquid-crystalline phase transition temperature of DMPC (as determined by ESR spectroscopy) were observed when the enzyme was present. These results have established that no significant incorporation of acetylcholinesterase into the hydrophobic region of the phospholipid bilayer is detectable at the protein: lipid ratios used in these experiments. Confirmation of these results was also obtained by differential scanning calorimetry. Interaction of the enzyme with the outermost region of the bilayer was established by trypsin digestion which indicated that as much as 25% of liposome-associated enzyme was removable and was, therefore, exposed to the outer surface. The results of this study have established that acetylcholinesterase associated with unilamellar DMPC liposomes was primarily entrapped within the aqueous compartment of the vesicles and was not present in the phospholipid bilayer.
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PMID:The topography of acetylcholinesterase in dimyristoylphosphatidylcholine liposomes. 254 25

About 5% of the total adenylate kinase activity in the rat forebrain was found in a subcellular fraction enriched in synaptic plasma membrane (SPM). The enzyme remained membrane bound after washing by 1M potassium acetate. It was resistant to trypsin digestion under conditions which destroyed 90% of acetylcholinesterase activity. The SPM enzyme was solubilized by 0.25% Triton X-100 resulting in a 4-fold increase in activity. Similar effects were observed when SPM was treated with phospholipases, melittin and trifluoperazine. These results suggest the occurrence of an adenylate kinase closely associated with SPM the activity of which can only be fully expressed by disturbances to the hydrophobic lipid bilayer. The enzyme can be seen as strategically located to play a role in regenerating ATP required for the manifold activities of the synaptic membrane.
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PMID:Evidence for a synaptic plasma membrane associated adenylate kinase in the rat brain. 255 31

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30-40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10-11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.
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PMID:Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system. 272 20

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.
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PMID:Multinuclear magnetic resonance studies on serine protease transition state analogues. 276 49

As a first step towards the identification and purification of the molecule(s) that are involved in cell contact-mediated tyrosine hydroxylase (TH) induction in cultures of bovine adrenal chromaffin cells, we have prepared plasma membranes (PM) from bovine adrenal medulla and tested their ability to mimick cell contact-mediated TH induction in low density chromaffin cultures. PM indeed induced TH in a manner similar to that observed in high density cultures. The maximal TH induction reached by PM corresponded to 69% of that of high density cultures, and half-maximal TH induction was obtained with 12 micrograms of PM per ml of medium. The induction of TH by PM was blocked by alpha-amanitin as observed in high density cultures. Since acetylcholinesterase was neither induced in high density nor in PM-treated low density cultures, an induction of TH as a result of a general increase in protein synthesis was excluded. The cell contact molecule(s) appear to be intrinsic membrane proteins. They were not removed by high or low salt extraction, but solubilized by 50 mM octylglucoside. They were resistant to 0.1% trypsin and heat denaturation but inactivated by 0.01% chymotrypsin. PM isolated from the adrenal cortex, kidney, and liver also induced TH in low density chromaffin cell cultures, although to a smaller extent than PM of the adrenal medulla. In contrast, muscle and erythrocyte PM were inactive. This shows that the cell contact molecule(s) are not restricted to the adrenal medulla, but are also present in some other but not all tissues.
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PMID:Selective induction of tyrosine hydroxylase by cell-cell contact in bovine adrenal chromaffin cells is mimicked by plasma membranes. 287 96


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