Enzyme activity biochemical assays

Active immobilized enzyme in biochemical assays using mass spectrometry... 

Recently, we have provided the first direct evidence for die existence of HCS activity in plant [10]. Indeed, we have partially purified and biochemically characterized HCS activity from pea leaves. The enzyme activity was assayed using D-[ H]Wotin and bacterial apo-BCCP (the biotinylated subunit of ACCase) from an E. coli mutant lacking in vitro HCS activity, as substrates. Several features of the reaction demonstrate that the plant HCS is responsible for the specific observed D-[ H]biotin incorporation (Table 1). First, we have verified that the reaction catalyzed by the plant HCS was dependent upon the presence of ATP, MgCl2 and apo-BCCP extract in the reaction medium (Table 1). Conditions for optimal catalytic activity and biochemical parameters of the plant enzyme were determined. HCS was active over a wide range of pH with an optimum between pH 7.5 and 8.5. 

Figure 1. Effect of temperature on stability and activity of Clostridium thermohydrosulfUriciun strain 39E amylopuUulanase. (a) TherW stability. The enzyme was placed in acetate buffer (SO mM, pH 6.0) with 5 mM CaC and preincubated at various temperatures for 30 min, and then residual amylopuUulanase activities were assayed, (b) Effect of heat on activi. The enzyme activity was assayed at various temperatures by the standard assay method (30 min incubation). (Reproduced with permission from Ref. 55. Copyright 1988, The Biochemical Society and Pordand Press, London.)...

The discussion above was concerned with the effects of solution conditions on enzyme activity, hence reaction velocity. Equally important for the purpose of assay design is the influence of specific solution conditions on the detection method being used. This latter topic is beyond the scope of the present text. Nevertheless, this is an important issue for screening scientists whose job is often to balance the needs of biochemical rigor and assay practicality in development of an HTS assay. An... 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

For biochemical assays, /iPLC allows direct quantification of substrates and products using a much-valued separation-based approach that allows development and optimization of challenging enzymatic assays faster and with fewer false positives. The separation-based approach employed by /iPI. C dramatically reduces assay development time from months to a few days. Since substrate and enzymatic products are separated prior to detection, /iPLC enables development of difficult assays, such as analyzing enzymes with low kinetic activities and enzymes that cannot be analyzed on existing platforms. 

Inhibiting the production of the Aft peptides represents the most direct approach to curtailing their potential to accumulate as amyloid plaques, by inhibiting either the ft-sec-retase at step 1 or the y-secretase at step 2. Because these are enzymatic reactions with measurable products, a biochemical assay using a purified enzyme preparation can be integrated into an HTS platform, facilitating the rapid evaluation of large numbers of compounds for inhibition of the enzymatic activity. 

A very useful complement to enzyme assays as described above is histochemical study, which can provide additional information [76]. In particular, because it is possible to measure the activity cell per cell, histochemistry permits, in the case of a heteroplasmic population of mitochondria, the detection of even a small number of affected cells, which may have remained undetected by biochemical assays. Spectacular images showing, side-by-side, cells endowed with either high or absent enzyme activity can be obtained. The limitation of the method is in part due to the few activities possibly measured (essentially complex IV, succinic dehydrogenase, and less specifically, ATPase and NADH reductase) and to the fact that it is poorly quantitative. Histochemical investigations are performed under selected conditions (e.g., substrate concentrations, pH), which often differ from those used for enzyme assays, thus possibly introducing discrepancy between the two approaches. 

While biochemical assays can provide receptor binding or enzyme inhibition information, they cannot determine how a compound behaves in a more complex cellular environment. Assays involving whole cells are required for this information. Compounds that show activity in a biochemical screen but fail in a cellular assay likely suffer from interference from the cell... 

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