Assay of the Enzymes

In another report, Michaelis-Menten parameters of ALP have been obtained using a one-shot Lineweaver-Burk (reciprocal) plot. This can be achieved by simultaneously measuring the conversion of 12 independent concentrations of the substrate (4-methylumbelliferyl phosphate) created on-chip. The enzyme was streptavidin-conjugated ALP that was linked to biotinylated phospholipid bilayers coated inside PDMS microchannels. The blue fluorescence of the enzymatic product, 7-hydroxy-4-methyl coumarin, was measured [ 1043]. The surface-bound enzyme was found to have a lower (sixfold) turnover rate than the free enzyme in solutions. After diffusion mixing between two streams (substrate and buffer) 

In another report, several acetylcholinesterase (AChE) inhibitors, including tacrine, edrophonium, tetramethyl- and tetraethyl-ammonium chloride, carbofu-ran, and eserine were assayed on a chip [1045]. AChE converted the substrate, acetylthiocholine, to thiocholine. This product reacted with coumarinylphenyl-maleimide (CPM) to form thiocholine-CPM (a thioether) for LIF detection. Since the acetonitrile solvent used to dissolve CPM inhibited AChE activity, the CPM solution was added after the enzymatic reaction [1045]. Another enzyme inhibition assay using a peptide substrate was performed on a PMMA chip [1046]. 

In one report, the activity of the HRP enzyme was studied by CL detection using the xanthine/xanthine oxidase (XOD)/luminol system. The enzymes were immobilized on glass beads trapped in a reaction chamber by weirs. It was found that when HRP was immobilized, a greater CL signal was generated than with free-solution HRP. However, when XOD (not HRP) was immobilized, enzymatic reaction products were not detectable. This was probably because the immobilized XOD enzyme had a low specific activity [721]. 

Enzyme kinetics were evaluated in a PDMS-glass chip using a continuous-flow system. A biotinylated enzyme (HRP or (5-galactosidase) was coupled to streptavidin-coated beads via the amide coupling of an aminocaproyl spacer. These beads (15.5 pm) were retained by a weir in the chip. The channel wall was passivated by 1 mg/mL BSA. The apparent enzyme kinetic parameters were evaluated using the Lilly-Homby model, as developed for the packed-bed enzymatic reactor systems. It was found that the apparent Michaelis constant (Km) approached the tme Km value of the free enzyme at zero-flow rate of a homogeneous reaction [845]. 

The activity of [i-galactosidasc (P-Gal) was studied on a quartz chip using a static micromixer to mix the enzyme and substrate on the ms time scale. Inhibition by phenylethyl-P-D-thio-galactoside was also studied [1048]. In another report, the enzyme P-Gal was assayed on a chip in which P-Gal would convert a substrate, resoruhn-P-D-galactopyranoside (RBG), to resoruhn to be detected fluorescently [1049]. By varying the substrate concentrations and monitoring the amount of resoruhn by LIF, Michaelis-Menten constants could be determined. In addition, the inhibition constants of phenylethyl-P-D-thiogalactoside, lactose, and p-hydroxymercuribenzoic acid to the enzyme P-Gal were determined [1049]. 

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Milstien S, Holtzman NA, Flynn ME, Thomas GH, Butler IJ, Kaufman S (1976) Hyper-phenylalaninemia due to dihydropteridine reductase deficiency. Assay of the enzyme in fibroblasts from affected infants, heterozygotes, and in normal amniotic fluid cells. J Pediatr 89 763-766... 

GDH activity in marine systems (Bidigare et al, 1982 Park et al, 1986). Assays of the enzyme in the reverse direction are virtually identical to those discussed below. 

In strictly diffusion-controlled reactions, i.e. at low efficiencies, simply spoken, only a small fraction of the total immobilized enzyme quantity is working. If in the course of operation actively working enzyme is destroyed, some other previously resting fraction may substitute to some extent. This is one reason why immobilized enzymes may erroneously appear to be more stable than free enzymes. To avoid such misinterpretation requires the assay of the enzyme activity in the absence of diffusional limitations. For industrial applications it is more useful to consider the enzyme s performance as indicated in Fig. 1, instead of just assessing its half-life. 

Assay of the enzyme activity is complicated by the fact that 7a-hydroxylation of cholesterol also may occur due to auto-oxidation or secondary to lipid preoxidation. In addition it is difficult to equilibrate exogenous cholesterol with endogenous microsomal cholesterol. 

Because of the difficulty of treating a number of the sphingolipidoses, clinical biochemists have been very interested in the development of simple diagnostic tests which can also be used for heterozygous individuals. Fortunately, the enzyme deficiency is the same in all tissues (Table 12.6). Thus, easily available materials can be used for the assay of the enzymes concerned in each disease. Such material includes leukocytes, cultured skin fibroblasts, serum and hair follicles. Urine and tears have also been used. In most cases the enzymes are stable to freezing so that samples may be shipped to diagnostic centres where automated assays have been developed for many of the determinations (e.g. Peters et ai, 1975 cf. Brady, 1978). 

When carboxylase was inactivated by phosphorylation for different periods of time, the polymeric form of the enzyme was progressively lost, as shown in Fig. 8. When the enzyme was inactivated for 3 minutes, the only detectable activity was in the protomers (17 to 22 S) and the intermediates (30 to 35 S). In this experiment, inactivation and sucrose density gradient centrifugation were carried out in the presence of 2 mM citrate, and the carboxylase assay was carried out in the presence of 10 mAf citrate. This experiment shows that phosphorylation and inactivation of carboxylase are accompanied by the conversion of polymers (45 S) into protomers and intermediate forms. It should be noted that loss of enzyme activity is not simply the result of the formation of the carboxylated form of the enzyme, E Biotin CO2 (Section III,A), since assay of the enzyme in the presence of 10 mAf citrate does not restore full activity, and inactivation occurs in the absence of CO2 (Fig. 5). 

To determine the poly(ADP-ribose) synthetase activity in the nuclear matrix, samples were incubated as above in 10 /uM [ P]-NAD in buffer A except that PMSF was omitted. For the assay of the enzyme activity in the subnuclear fractions, 25 jug DNA ml, 25 jug histone Hi ml, and 60 jug DNase I ml were additionally included. 

Morre et al. (1970) detected the synthesis of PC from CDP-choline in extracts from onion stem. The activity was associated with a particulate fraction. Devor and Mudd (1971b) found that the 100,000-g pellet from spinach leaves had the highest total amount and specific activity of this enzyme. The metal ion requirement could be satisfied by Mn + (saturating at 0.3 mM) or Mg + (saturating at 13 mM). The for CDP-choline was 10 pM, and the pH optimum was 7.5-8.0. Only modest stimulation could be achieved by the addition of DG. Johnson and Kende (1971) measured the enzyme in particulate fractions (44,000-g pellet) from barley (Hordeum vulgare L.) aleurone layers and found the activity greatly stimulated when the aleurone layers were exposed to gibberellin before extraction and assay of the enzyme. 

In 1978 Fischbein et al, described 5 patients with a myo-adenylate deaminase (MAD) deficiency, which were detected during a histochemical screening of 250 consecutive muscle biopsies. The deficiency was confirmed by a biochemical assay of the enzyme. Clinically three of the patients complained of muscle cramping, in one case associated with postexercise fatigue. CK was only moderately elevated, the myogram showed minor abnormalities, in muscle there were histologically no or only slight alterations. These three patients didn t show any muscle weakness or atrophy. 

When arylazido- 3-alanine ATP containing P in the y phosphate is incubated with F, (ATPase) in the dark, there is a sizable turnover of the enzyme. Within 15 sec, in excess of 40% of the added 2 mmoles of the analog one is hydrolyzed by 6.37 /ng of Fi (ATPase). Assay of the enzyme under these conditions, i.e., in the absence of a nucleotide re-... 

The dehydrogenase of E. coli has been shown to be inactive in the absence of divalent cations, and indeed the maximal actiiaty of the enzyme from annelid eggs or E. coW requires a relatively laxge amount of Mg++ or Ca++ (0.02 M). Assays of the enzyme have frequently been unsatisfactory because optimal ionic conditions were not provided. The enzyme is inhibited by heavy metals, such as Cu++. Thus the activity of this critical enzyme in glucose utilization is dependent on the interactions of protein, coenzyme, substrate, and divalent cations, and is sensitive to numerous inhibitory substances, some of which have been demonstrated to be active as chemotherapeutic agents. 

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