Studies on the Selective Chemical Inhibition by Urea of Alkaline Phosphatase Isoenzymes in the Reaction Course

Abstract

Urea inhibits the activity of alkaline phosphatase during the reaction course. The inactivation is progressively stronger for the placental, intestinal and renal subforms. Influence of reaction temperature, pH, type and molarity of buffer, magnesium chloride, albumin and enzyme concentration on the inactivation mechanism is evaluated. In all experimental conditions the process follows pseudofirst-order kinetics and the inactivation profiles are distinct and typical for each enzymatic subform. With a simple graphical analysis, a single inactivation curve in controlled experimental conditions, allows the identification of each isoenzyme from the slope and the calculation of the respective fractional amount from the intercept of the time-activity plot.

Alkaline Phosphatase Expression in Human Chorionic Villi

“…3). In fact, kinetic pattern is typical of a binary mixture of A1P components with Kobs value (exponential 1 = 3.8 X 10"2; exponential 2 = 4.2 X 10"1 ) as previously demonstrated for renal, placental and neu trophil A1P [14,17].…”

“…The urea selective inhibition was investigated in duplicate with 3.7 mol/l urea, by continuous record ings at 37 °C for 15 min. Analysis of time activity curves was performed as previously described [14].…”

Time-Resolved Fractionation of Bone and Liver Alkaline Phosphatase Activities with a ‘Peeling-Off’ Method

“…The graphic analysis [21] shows that the discrimination power of one procedure relies upon a satisfactory diversity of the apparent inactivation constants (kapp), so that the larger the difference the more evi dent the cut-off point appears. The separa tion of the hepatic from the intestinal sub form (kapp = 0.035 min-1) could be obtained by extending the reading time up to 15 min, while this procedure cannot be practically employed to part off the hepatic from the placental enzyme, whose kapp is 0.016 min-1, as previously reported [16]. In 10 cases …”

“…The computer-as sisted estimate of the fractional amounts present in isoenzymatic mixtures was performed as described [16]. We have experienced a centrifugal analyzer (CentrifiChem; Union Carbide, USA) which permits to perform 25 inactivation curves simultaneously.…”

“…After studying the influence of different experimental conditions on the kinetic frac tionation of mono-, bi-and tri-component mixtures of purified enzymes [16], we have however considered this type of procedure worthy of further efforts. An application of our procedure to the differentiation of neu trophil AP isoenzymes has already appeared [17], In this regard, the analytical perfor mance of the kinetic approach, based upon urea-selective inactivation, is here evaluated with the aid of a 'peeling-off' graphic method and applied to the quantitation of the bone and liver subforms in human sera and other biological samples.…”