N-Ethylmaleimide inhibition

NOCICEPTIVE BEHAVIOR INDUCED BY THE ENDOGENOUS OPIOID PEPTIDES DYNORPHINS IN UNINJURED MICE EVIDENCE WITH INTRATHECAL N-ETHYLMALEIMIDE INHIBITING DYNORPHIN DEGRADATION... 

Inhibition patterns of [ N]ammonia accumulation by strains JL907 and JB822 (GDH") are shown in Figure 4. It is clear that assimilation plays an important role in [ N] ammonia accumulation in these strains. Strain JB822 is resistant to methionine sulfoximine due to a defect in methionine transport (data not shown). The inhibition by metabolic inhibitors (Figure 4) was not unexpected, since assimilation and all possible accumulation mechanisms (Table II) are energy-dependent. It is presently unclear whether N-ethylmaleimide inhibition was due to inactivation of a sulfhydryl-containing surface receptor or to inhibition of electron transport. 

FIGURE 5 Plots of the apparent pseudo first-order rate constants for the N-ethylmaleimide inhibition of the condensation reaction using 16 0 CoA ( ), 6,9-18 2 CoA (o), and 6,9,12-18 3 CoA (A) as substrates. 

The results in Table 6 were obtained when both the overall rates of chain elongation and the condensation reaction were measured with 16 0 CoA, 6,9-18 2 CoA and 6,9,12-18 3 CoA as substrates The 8-hydroxy acyl-CoA dehydrase reaction was assayed only with the CoA derivatives of DL-8-hydroxy-stearic acid (8-OH-18 0 CoA) and DL-8-hydroxy-8,11-eicosadienoic acid (8-OH-8,11-20 2 CoA). The 2-trans-enoyl-CoA reductase reaction was assayed with the CoA derivatives of 2-trans-octadecenoic acid (2 trans-18 1 CoA) and 2-trans-8,ll-eicosatrienoic acid (2-trans-8,11-20 3 CoA) All reactions were assayed with the microsomes from rats raised on both a chow diet and a fat-free diet. Both the rates of the condensation reaction and the overall chain elongation reaction were depressed when rats were fed a chow diet These depressed rates of conversion were more pronounced when 16 0 CoA was used as substrate than when 6,9-18 2 CoA or 6,9,12-18 3 CoA were used as substrates. These results support our N-ethylmaleimide inhibition studies and are consistent with the concept that rat liver microsomes contain at least two different condensing enzymes. One condensing enzyme would preferentially utilize saturated substrates while another would act on unsaturated substrates. 

Step 7 Hydrolysis of ATP by NSF is essential for fusion, a process that can be inhibited by NEM N-ethylmaleimide). Gertain other proteins and calcium are also required. 

On the other hand, the use of a-cyclodextrin decreased the rate of the reaction. This inhibition was explained by the fact that the relatively smaller cavity can only accommodate the binding of cyclopentadiene, leaving no room for the dienophile. Similar results were observed between the reaction of cyclopentadiene and acrylonitrile. The reaction between hydroxymethylanthracene and N-ethylmaleimide in water at 45°C has a second-order rate constant over 200 times larger than in acetonitrile (Eq. 12.2). In this case, the P-cyclodextrin became an inhibitor rather than an activator due to the even larger transition state, which cannot fit into its cavity. A slight deactivation was also observed with a salting-in salt solution (e.g., quanidinium chloride aqueous solution). 

Table II shows that UDP-pyridoxal had a similar inhibitory effect on red beet glucan synthase. It inhibited activity at much lower concentrations than other covalent modification reagents, such as N-ethylmaleimide (cysteine), phenylglyoxal (arginine) and formaldehyde (lysine). UDP-pyridoxal had an I50 that is 62-fold lower than formaldehyde.

Another successful example of such guest design is the Diels-Alder reaction markedly accelerated by the cyclodextrin inclusion. As shown in Table XXIV, /J-cyclodextrin accelerates the addition of a small dienophile to cyclopentadiene, but inhibits that of N-ethylmaleimide to anthracene-9-carbinol (117). Thus, the guest design is a really helpful concept for the remarkable catalysis. However, there seems to be some limitation in the choice of reactions, if cyclodextrins have no special functional group for the... 

The enzyme activity is significantly inhibited by Tiron and 8-hydroxyquinoline, but not by a,a -dipyridyl and o-phenanthroline. The addition of thiol compounds such as cysteine, 2-mercaptoethanol and glutathione, and thiol inhibitors such as p-chloromercuribenzoate, N-ethylmaleimide and HgCl2 also markedly decreases the enzyme activity. The Michaelis constants of the enzyme are as follows 2-nitropropane (2.13 x 10-2M), nitroethane (2.43 x 10-2M), 3-nitro-2-pentanol (6.8 x 10 3M), 1-nitropropane (2.56 x 10-2 M) and oxygen (3.63 x 10 4M with 2-nitropropane)199. ... 

Martin (1971) found that the transport of choline across the red cell membrane was inhibited by N-ethylmaleimide (NEM), a sulphydryl reagent which covalently modifies cysteine residues in proteins. The extent of the inhibition depended on... 

V. N-Ethylmaleimide-Induced Nociceptive Behavior Mediated Through Inhibition of Dynorphin Degradation... 

Protease Classification. In order to rationally design an inhibitor for a protease it is first necessary to place it into one of four families of proteases (see Table V). For a new enzyme, a study of its inhibition profile with a series of general protease inhibitors is sufficient to classify it into one of the four families. The inhibitors usually used are diiso-propylphosphofluoridate (DFP) or phenylmethane sulfonyl fluoride (PMSF) for serine proteases, 1,10-phenanthroline for metalloproteases, thiol reagents such as iodoacetate or N-ethylmaleimide for thiol proteases, and pepstatin or diazo compounds such as diazoacetyl-norleucine methyl ester for carboxyl proteases. 

Both are probably SH enzymes, being inhibited by I2, Ag", p-chloro-mercuribenzoate and N-chlorosuccinimide, although not by iodoacetate or N-ethylmaleimide (85). Mercaptoethanol is commonly used in purification (83). The enzymes are also inhibited by Co ", Mn ", and Fe ", but chelating agents are without effect (85). Neutral salts such as NaCl also inhibit the mustard enzyme (87). 

The only cofactor required for this condensation is a divalent cation, either Mg or Mn ", and the activity can be inhibited by iodoacetamide, N-ethylmaleimide and p-hydroxymercuribenzoate. Cell-free extracts catalyzing phytoene formation have also been isolated from pea fruits spinach leaves Mycobacterium, Halobacterium cutirub-... 

An interesting contribution of steric effects to solvation is the addition of benzenethiolate ions to N-ethylmaleimide in 95% EtOH (52). The reactivity ratio of the 4-H 4-Me 2-Me 2,6-Me2 derivatives is 1 2.16 1.6 1.6 thus, a small steric retardation is superimposed on a small electronic acceleration. However, the 2-t-Bu derivative is 8.7 times faster than its 4-t-Bu isomer. This finding was ascribed to a rate-enhancing steric inhibition to solvation of the anion, which raises its reactivity by more than the reactivity decrease due to crowding in the transition state. 

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