On the Mechanism of Inhibition of Intestinal Alkaline Phosphatase by L-Phenylalanine
نویسندگان
چکیده
The degree of inhibition of rat intestinal alkaline phosphatase by L-phenylalanine was highly pa-dependent and varied from 0 to 66% within a pH range of ‘7.8 to 10.4, exhibiting a peak at pH 9.2 and 8.7 for phenylphosphate and /3glycerophosphate, respectively. Vm,, was also a function of pH with and without the inhibitor. Rat intestinal alkaline phosphatase exhibited maximum enzyme activity at pH 9.8 and 8.8 with substrates, phenylphosphate and P-glycerophosphate, respectively, in presence of the noninhibitor, n-phenylalanine. The corresponding pH optima in the presence of L-phenylalanine inhibitor were 10.2 and 9.3, respectively. This shift in optimum pH by the inhibitor was observed in systems containing carbonatebicarbonate or borate buffers. The Michaelis constant was pH-dependent. The Dixon plot (pK, with respect to pH) showed one discontinuity at pH 8.6 for the free enzyme and another at pH 9.6 for the enzyme-phenylphosphate complex. The values for the energy of activation for the enzymecatalyzed hydrolysis of phenylphosphate with and without L-phenylalanine were 18,000 and 6,000 calories per mole, respectively. The inhibition was greatly dependent on substrate and inhibitor concentrations, and was of the “uncompetitive” type, because the double reciprocal plots of velocity and substrate concentrations in the presence of four different concentrations of L-phenylalanine were all straight lines parallel to those obtained without the inhibitor, in both the cases of phenylphosphate and P-glycerophosphate. At this time, the kinetic data are interpreted as indicating either the formation of a thermodynamically stable enzymeinhibitor-substrate complex which, in effect, reduces the concentration of enzyme-substrate complex available to decompose into products or the production of a weakly dissociable enzyme-inhibitor-substrate complex. These interpretations are relevant to the explanation of the stereospecific, organ-specific inhibition of rat intestinal alkaline phosphatase by L-phenylalanine.
منابع مشابه
Quantitative method for determining serum alkaline phosphatase isoenzyme activity: estimation of intestinal component.
Intestinal alkaline phosphatase activity was measured using levamisole inhibition, and results were compared with a previously reported method using L-phenylalanine. Sixty two per cent intestinal, 39% placental, and 1.3% of either bone or liver alkaline phosphatase activity remained when alkaline phosphatase activity was inhibited in a 2-amino-2-methyl-1-propanol (AMP) buffer reagent system wit...
متن کاملOn the mechanism of inhibition of intestinal alkaline phosphatase by L-phenylalanine. I. Kinetic studies.
The degree of inhibition of rat intestinal alkaline phosphatase by L-phenylalanine was highly pa-dependent and varied from 0 to 66% within a pH range of ‘7.8 to 10.4, exhibiting a peak at pH 9.2 and 8.7 for phenylphosphate and /3glycerophosphate, respectively. Vm,, was also a function of pH with and without the inhibitor. Rat intestinal alkaline phosphatase exhibited maximum enzyme activity at ...
متن کاملSerum Alkaline Phosphatase
phatase may reflect enzyme contributions from bone, liver, and intestine. We have investigated serum alkaline phosphatases in two siblings with hypophosphatasia. After administration of long-chain triglycerides, the major alkaline phosphatase component of their sera was shown to be of intestinal origin on the basis of inhibition by l-phenylalanine. Starch block electrophoresis suggested that th...
متن کاملIntestinal alkaline phosphatase in bile: evidence for an enterohepatic circulation.
The isoenzyme characteristics of alkaline phosphatase in human bile obtained from patients with gallstones have been determined by means of electrophoresis on polyacrylamide gel, both before and after preincubation with neuraminidase, and also by means of inhibition tests with heat, urea, and L-phenylalanine. Bile alkaline phosphatase is shown to be partly secreted by the liver cell, but partly...
متن کاملL-Phenylalanine inhibition of human alkaline phosphatases with p-nitrophenyl phosphate as substrate.
With p-nitrophenyl phosphate as the substrate, there reportedly is no organ-specific inhibition of alkaline phosphatase (EC 3.1.3.1) activity by L-phenylalanine. However, we found that at pH 10.0, with p-nitrophenyl phosphate as the substrate, L-phenylalanine obviously inhibits the alkaline phosphatase isoenzyme from human placenta, whereas there is little if any inhibition of the isoenzyme fro...
متن کامل