Assays competitive inhibition enzyme

Competitive Inhibition Enzyme Assays. Estimates of antibody-lysozyme dissociation constants can be obtained by taking advantage of the fact that most monoclonal antibodies efficiently inhibit enzymatic activity.3 5 The combining site of HyHEL-10, which is presented as an example, also has been demonstrated by X-ray crystallography to overlap a portion of the catalytic site of lysozyme.7 A constant concentration of lysozyme is incubated with varying amounts of antibody, and amounts of free (unbound) lysozyme molecules are estimated by the proportion of catalytic activity remaining. The assay assumes that the addition of Micrococcus lysodeikticus cell walls and concurrent dilution of the antibody-antigen mixture do not disturb the equilibrium. 

Fig. 1. Effect of HyHEL-10 on M. lysodeikticus lysis activity of chicken lysozyme at 0.1 nM concentration using the competitive inhibition enzyme assay described in the text (a) Raw data with varying concentrations of antibody and the substrate-only control, (b) Plot of antibody concentration versus decrease in absorbance at 450 nm per minute at 0.1 nAf enzyme (unconected for substrate-only control).

Muldoon et al. developed a monoclonal-based competitive inhibition enzyme-linked immunosorbent assay (cELISA) for sulfadimethoxine. The group compared... 

Reversible, non-competitive inhibition of polymerase is also afforded by a series of N-benzoyl pyrrolidines. Substitution on the benzoyl moiety with a para-trifluoromethyl group is optimal in this series. Bulky, hydrophobic groups at the 2-position of the pyrrolidine ring increase activity, and the 5-position tolerates a wide range of substituents, indicative of a solvent exposed portion of the inhibitor. Compound (+)-38, containing a 2-thienyl moiety at the 5-position, has an IC50 of 190 nM in the enzyme assay while its enantiomer is almost 100-fold less active [83]. 

Any compounds (other than the physiologically relevant substrate) that can serve as substrates for a particular enzyme. Alternative substrates compete with the natural substrate and with each other for access to the enzyme s active site. Thus, if one is utilizing an assay that measures the production of the true substrate, then the presence of the alternative substrate will result in competitive inhibition relative to the true substrate. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

Table-TV shows the effect ot fhe LMW fraction on the activity of some of these enzymes in vitro. Maltase, lactase and invertase were competitively inhibited at a concentration of 10 mg/ ml. When the effectsof a range of concentrations (2.5-20 mg/ml) of the LMW fraction were studied, it was revealed that the inhibition was not of the pure competitive type. Table V shows the effect of the HMW fraction. Low concentrations had to be used in the assays, as the intense brown color of this fraction interfered with the spectrophotometric measurements. In spite of this a strong competitive inhibition of lactase and of invertase was found. Maltase was also inhibited, and, to a lesser extent, even trehalase. a-Amylase from saliva was not affected at the concentration tested.

For competitive, reversible enzyme inhibition, the lowest measurable IC50 value is half of the enzyme concentration used in the assay (Cheng and Prusoff, 1973). From a practical view, kcJKM values of 104 M 1 s 1 and above are desirable for inhibitor profiling assays. With an enzyme-substrate pair characterized by a kcJKM value of 104 M 1 s, an assay can usually be run with a protease concentration in the single-digit nanomolar range in an automated setting. 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

An enzyme catalyzing the reaction S P is competitively inhibited by I. The assay is based on the incorporation of label from radioactive S into P. = 2.3xio M and V max = 290 nmoles/min under standard assay conditions. -Ki = 2.3 X 10 M. The specific activity of the labeled S is 4.5 x lO CPM//i.mole. (a) What is the observed velocity in terms of CPM incorporated into P per minute when [S] = 2.3 X10 M (b) What would the observed rate be (CPM/min) if a fivefold excess of unlabeled S were added (c) What would the observed rate be (CPM/min) if a fivefold excess of I were added ... 

A second type of EMIT has been developed using the enzyme malate dehydrogenase as the enzymatic label. Research has shown that thyroxine competitively inhibits malate dehydrogenase. A conjugate prepared with thyroxine covalently bound close to the enzyme s active site shows very low specific activity that can be restored by binding of the thyroxine to arcP -thyroxine antibody. In this very specific assay for thyroxine, enzyme activity increases upon antibody binding, so that in a competitive assay for free thyroxine, activity decreases with increasing free thyroxine concentration. 

It has been shown that a 30% inhibition could be obtained in CPT activity when ceramides were directly added to the assay mixture at 50 pM concentrations (Bladergroen et al., 1999). Since this inhibition of 30% was less than the 64% obtained when cells were incubated directly, it would indicate competitive inhibition was not the only mechanism. In the present work, we found similar results with lung microsomal fraction, that is, with an increase in the incubation time with ceramides, the inhibition of the enzyme activity increases. Therefore, we support the observations by Bladergroen et al. (1999) that ceramide inhibition of CPT activity may be only partially through direct competitive inhibition with DAG ceramide may act through interaction with other CPT enzyme inhibitors present in the microsomal fraction. 

Very recently we raised three monoclonal antibodies against human liver Mn-SOD. The epitope of one of these antibodies was found to be a COOH-terminal peptide, as judged by competitive inhibition assay using synthetic peptides (K4). Using this antibody we developed an ELISA method and found that the enzyme is also present in human serum. Measurement of the serum immunoreactive Mn-SOD protein levels in various diseases revealed that the enzyme levels are... 

Irreversible, covalent inhibition has been explored as an approach to overcome competition by ATP and ADP. Such competition is thought to contribute to the large drop in potency often seen on moving from isolated enzyme assays, typically at around 10 pM ATP, into cells where are ATP and ADP each are present around mM concentrations. 

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