Alkaline phosphatase, activation volume

The treated cells were placed into the electrochemical chambers and the substrate PAPP was added to a 1 mg/ml final concentration at a total volume of 100 nL. Alkaline phosphatase activity was measured by monitored the PAP oxidation current. The electrochemical chips are disposable and were replaced every experiment. 

Fig. 12.4 HT-29 colon cancer cells response to BA, AN-7, and AN-9. Amperometiic response curves for monitoring of alkaline phosphatase activity using the electrochemical array chip. The HT-29 colon cancer cells were exposed to the differentiation agents Butyric acid (2.5 mM), AN-7 and AN-9 (50 pM). The HT-29 cells with the substrate PAPP were placed into the 100 nL volume electrochemical chambers on the chip. Current was measured using the amperometric technique at 220 mV...

In practice, using a normal chemistry panel and complete blood count it is not unusual to have 30 potential covariates, everything from sodium ion concentration to alkaline phosphatase activity. Early in PopPK analyses it was not unusual to screen every single covariate for their impact on the model. But a model might end up having a volume of distribution as a function of chloride ion concentration or clearance that is a function of glucose concentration. Physiologically, these covariates are nonsensical. Ideally at the end of model... 

A rotating enzyme-immobilized reactor and a flat pH electrode were incorporated into a sealed cell for use under continuous-flow/stopped-flow (SF) operation for the rapid determination of penicillins G and V in tablets and injectables [50]. A co-immobilization in a rotating bioreactor and amperometric detector resulted in a sensitive system for determination of succinylcholine and acetylcholine in pharmaceutical preparations [51]. A tandem system incorporating two rotating bioreactors into a continuous-flow/SF sample/reagentprocessing setup was apphed for the determination of alkaline phosphatase activity in serum samples [52]. By functional combination of the SF and flow-injection analysis (FIA), an automated micro apparatus was constructed resulting in significant reduction of the injection volumes of enzyme and substrate [53]. SF/continuous flow methods were apphed to acquire kinetic information also [54, 55]. 

The comparative toxicity of retinoids and retinamides have also been studied by Sani and Meeks (1982). On the basis of clinical signs of toxicity, lethality, and histopathological findings in rats and mice, the toxicity of the retinoids and retinamides were ranked in the order tretinoin > 2-hydroxypropylretinamide > isotretinoin > 2-hydroxyethylretinamide > ethylretinamide > 3-hydroxypropyl-retinamide > 4-hydroxyphenylretinamide. Treatment with the retinoids (doses not stated) caused an anemia characterized by erythrocytopenia and decreases in hemoglobin concentration, packed-cell volume, and hematocrit. Dose-dependent increases in serum alkaline phosphatase activity and decreases in serum albumin were also observed. 

Escherichia coli can he adapted to grow on PPi as the sole source of phosphorus in this situation the cells are dependent upon intracellular inorganic pyrophosphatase activity to make Pi available. To avoid possible confusion from the inducible alkaline phosphatase of E. coli, which also has pyrophosphatase activity (see Chapter 17, by Reid and Wilson, this volume), all mutant isolation studies began with an E coli strain unable to synthesize this inducible protein. 

Reaction mixtures contained in a volume of 0.5 ml 50 jimoles HEPES pH 7.0, 0.5 pmoles CoCI2, 45 hq KOO-8-phosphate phosphatase or 5 / g alkaline phosphatase, and 0.5 mg of the appropriate compound, incubated at 35°C. Data for both enzymes are expressed as specific activities (pmoles per min per mg protein) under the above conditions of incubation. 

Measurement of Alkaline Phosphatase (ALP) Activity. Although a multitude of methods are being used to measure peroxidase, most workers use the following method to measure ALP. The reaction utilizes 1 mg of p-nitrophenylphosphate (Sigma) per milliliter in 1 M diethanolamine buffer, pH 9.8. The reaction is stopped by addition of i volume of 2 M NaOH. The yellow p-nitrophenol is measured at 405 nm. 

The product of the fourth reaction absorbs strongly at 520 nm, with a molar absorptivity of 2.3 x 104Af em. Following a 10-min incubation, 100 amol (1 amol = 10-18 mol) of alkaline phosphatase in 0.100-mL total volume hydrolyzes 0.1 mill of the FADP (0.5% of the initial saturating substrate concentration of 20 mM), and converts 12% of the originally 100 nm apo-D-amino acid oxidase to the active holoenzyme. 

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