Alkaline phosphatase phenylalanine

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Not all inhibitors fall into either of these two classes but some show much more complex effects. An uncompetitive inhibitor is defined as one that results in a parallel decrease in the maximum velocity and the Km value (Figure 8.8). The basic mode of action of such an inhibitor is to bind only to the enzyme-substrate complex and not to the free enzyme and so it reduces the rate of formation of products. Alkaline phosphatase (EC 3.1.3.1) extracted from rat intestine is inhibited by L-phenylalanine in such a manner. 

Fiqtire 3.5 (a) Competitive inhibition inhibitor and substrate compete for the same binding site. For example, indole, phenol, and benzene bind in the binding pocket of chymotrypsin and inhibit the hydrolysis of derivatives of tryptophan, tyrosine, and / phenylalanine, (b) Noncompetitive inhibition inhibitor and substrate bind simultaneously to the enzyme. An example is the inhibition of fructose 1,6-diphosphatase by AMP. This type of inhibition is very common with multisubstrate enzymes. A rare example of / uncompetitive inhibition of a single-substrate enzyme is the inhibition of alkaline phosphatase by L-phenylalanine. This enzyme is composed of two identical subunitjs, so presumably the phenylalanine binds at one site and the substrate at the other. [From N. K. Ghosh and W. H. Fishman, J. Biol. Chem. 241, 2516 (1966) see also M. Caswell and M. Caplow, Biochemistry 19, 2907 (1980). 

The relationship between the various tissue alkaline phosphatases has been under discussion for many years (24). Bodansky established that inhibition by bile acids could be used to distinguish between intestinal and bone or kidney isoenzymes (25). The organ-specific behavior of rat tissue phosphatases toward a variety of compounds was investigated by Fishman (26). Of particular importance was the observation that l-phenylalanine is a stereospecific inhibitor for the intestinal isoenzyme (27). Immunochemical (28, 29) and electrophoretic techniques (30, 31) have shown that there are also physical differences between the tissue phosphatases. It is not yet clear what the precise nature of these differences is (32), although in part it results from a variability in sialic acid content. 

Heterogeneous phosphorylation is often a problem when kinases are expressed in insect cells. Multiple approaches have been used to solve this problem. Proteins have been completely dephosphorylated by incubation with A protein phosphatase or alkaline phosphatase [38, 39, 56]. Ion exchange and isoelectric focusing chromatography have been used to separate proteins with multiple phosphorylation states. An y-aminophenyl ATP-sepharose column was used to separate different phosphory-lated states of human c-Src [34]. Alternatively, serine/threonine or tyrosine phosphorylation sites can be mutated to alanine or phenylalanine, respectively [42]. For tyrosine kinases with multiple autophosphorylation sites, the active site aspartic acid can be mutated to an asparagine, creating a kinase dead mutant [57]. 

A biochemical evalution of human alkaline phosphatase is postponed until the above considerations have been presented. In our view, the most reasonable analytical approach is based on the measurement of L-phenyl-alanine-sensitive and -insensitive moieties along with their respective heat stabilities. To this may be added information gathered from starch-gel electrophoresis with native and heated serum and from the presence of L-phenylalanine-sensitive bands on the gels following electrophoresis. Experiments of a different type can be included, in which the serum is incubated with neuraminidase and susceptibility of the glycoprotein is established following electrophoresis. Finally, the data on L-phenyl-alanine inhibition of heat-sensitive and -insensitive moieties appear to make sense, if the population of normal subjects is divided into one with the slow-moving intestinal band and one without it. It is from this consideration and other indirect and direct inferences that the intestine is... 

This color reaction has now provided measurements of total alkaline phosphatase and the L-phenylalanine-sensitive isoenzyme. 

From the biochemical point of view, there is merit in measuring the moiety of the serum alkaline phosphatase activity that is inhibited by L-phenylalanine. This moiety is henceforth referred to as LPSAP, L-phenylalanine-sensitive alkaline phosphatase. The conditions to be chosen should provide the maximum expression of LPSAP and the extent of inhibition of intestine and placenta should be as great as possible at a concentration of L-phenylalanine that does not at the same time inhibit... 

As reported earlier (F16, G8), placenta and intestine are two sources of alkaline phosphatase that are equally sensitive to L-phenylalanine. As can be expected, gastric and duodenal contents contain LPSAP as well. Preparations made so far from the following tissues contain much smaller amounts of LPSAP liver, bone, kidney, lung, and spleen, usually of the order of 0-15% (Table 4). 

L-phenylalanine, the percent inhibitions of human intestinal liver and bone alkaline phosphatases are 77, 8, and 10 (Table 4), respectively. 

An attempt was made to find conditions in which human intestinal alkaline phosphatase would exhibit L-phenylalanine inhibition higher than 77%, by changing the substrate and inhibitor concentrations. The results in Table 5 illustrate that the employment of higher inhib-... 

Sensitivity of Alkaline Phosphatase op Tissue and Body Fluids to l-Phenylalanine ... 

Fig. 4. Differential AutoAnalyzer alkaline phosphatase tracings. In each pair of peaks the tracings of the line containing L-phenylalanine precedes the one containing D-phenylalanine in the buffered substrate [according to Green et al. (G16)].

Fia. 6. AutoAnalyzer Manifold for L-phenylalanine-sensitive alkaline phosphatase (F8). Waste lines 13 and 14 are both 0.90 in. and line 15 is 0.073 in. 

Although a considerable literature records numerous studies in this field (A19, B17, C12, J3, L9, P16, S4, S5), many have been performed on preparations of tissues from other than human sources. In conformity with the subject of this chapter and to avoid species differences, most attention will be directed to human tissue alkaline phosphatases and in particular their variants. The stereospecific L-phenylalanine inhibition has provided the impetus to study its molecular mechanism, which necessarily requires an understanding of the mechanism of catalysis. It is expected that discovery of other stereospecific inhibitors will follow and that they may have even greater utility than L-phenylalanine. However, since it is the first such unique inhibitor, this section of the chapter will receive extensive treatment after a consideration of some basic kinetic information. 

Fia. 7. pH optima for intestinal and placental alkaline phosphatases (R) values in the absence of phenylalanine (D) in the presence of 0.005Af n-phenylalanine, and (L) in the presence of 0.005 Af L-phenylalanine. The substrate concentration was 0.018 Af phenyl phosphate. The triangles represent the intestine, and the circles represent human placenta. 

Amino acids have been studied in relation to alkaline phosphatase, and organ differences have been observed by Bodansky (B30) and Fishman (F13). Inhibition is competitive in nature. The most recent study on the influence of L-histidine demonstrates alteration in the pH optimum (B33) of hydrolysis. Moreover, a survey of amino acid inhibitors (Fll) has produced (F13) the unique stereospecific uncompetitive inhibitor, l-phenylalanine, which is discussed in detail in section 3.1.6. 

The discovery of the stereospecific inhibitor, L-phenylalanine, arose from a systematic study of rat tissue alkaline phosphatases (Fll) and from investigation of human intestinal and placental enzyme preparations (F13, F16). 

Certain kinetic features of L-phenylalanine inhibition will now be described for the purified human intestinal and placental preparations of alkaline phosphatase. In experiments on the effect of pH, Ghosh and Fishman (G5) observed that the degree of stereospecific L-phenylalanine inhibition of alkaline phosphatase from rat or human intestine and from human placenta is highly pH-dependent. Rat or human intestinal alkaline phosphatase exhibited maximum inhibition at pH 9.2 with phenyl phosphate as substrate, whereas the human placental alkaline phosphatase had a peak at pH 9.6 (Fig. 10). 

Fig. 10. Inhibition by L-phenylalanine as a function of pH. Triangles represent human intestinal and circles represent human placental alkaline phosphatase...

The L-phenylalanine inhibition of rat (G5) or of (Fig. 12) human intestinal alkaline phosphatase and of human placental (G6) enzyme is of the uncompetitive type, because the double reciprocal plots of velocity and substrate concentration were all straight lines parallel to those obtained without the inhibitor. Consequently, the extent of the inhibition was greatly dependent on substrate (Fig. 11) and inhibitor concentrations (Fig. 10). Detailed studies have appeared elsewhere (G5). 

Determination op fca op Intestinal Alkaline Phosphatase in the Presence op d- and l-Phenylalanine... 

Fig. 13. Representation of the formation of an EIS complex during alkaline phosphatase inhibition by L-phenylalanine. Compare with Fig. 9.

Starch-gel electrophoresis of the alkaline phosphatase in the butanol extracts of leukocytes revealed three variants of the enzyme. Peacock et al. (PI) have devised a method for leukocyte alkaline phosphatase assay. An additional variant was detected in blood leukocytes of leukemia patients treated with 6-mercaptopurine (RIO). Robinson and Pierce (R7) indicated that there might be a fundamental difference in molecular structure of the human serum alkaline phosphatase proteins because serum alkaline phosphatase, when incubated with neuraminidase prior to electrophoresis, demonstrated reduced anodal migration of those isoenzymes that are not L-phenylalanine-sensitive. L-Phenylalanine-sensi-tive enzyme of intestinal origin was found to be neuraminidase-resistant. 

Much attention has been devoted in this laboratory to the starch-gel electrophoretic studies of various human tissue alkaline phosphatases, especially those intestinal and placental alkaline phosphatase isoenzymes that undergo stereospecific inhibition by L-phenylalanine and not by its... 

Fig. 23. Demonstration of heterogeneity of L-phenylalanine-sensitive serum alkaline phosphatase in a patient with alcoholic cirrhosis [according to Kreisher et al. (K25)].

It is now possible to demonstrate [after electrophoresis for 18 hours at 5°C (B6)] the location of L-phenylalanine-sensitive alkaline phosphatase in gels directly by a postcoupling technique developed by Inglis and Fishman (Fig. 24) the simultaneous coupling reaction in the... 

The L-phenylalanine-sensitive component of serum alkaline phosphatase exhibits heat sensitivity that on occasion is far below or far above the expected heat sensitivity for intestine. For this reason, it is... 

The tissue preparations were diluted (1 12) in heat-inactivated sera (1 hour at 55°C), and incubated in 0.02 M Veronal buffer (pH 9.8) containing 0.018 M disodium phenyl phosphate for 2 hours at 37°C. Phenol was measured via a diazo coupling procedure. Conditions for heat-inactivating the tissue enzymes were 16 minutes at 55°C. By subtracting from the total activity the intestinal component, which is measured by n-phenylalanine sensitivity, one obtains the sum of the activities of liver and bone. The ratio of the two was computed from the heat inactivation minus that attributed to intestine, employing 91.2% heat inactivation to represent 100% bone and 51.4% heat inactivation indicating all liver. In this way one arrives at values for bone, liver, and intestinal alkaline phosphatase. 

Sites of alkaline phosphatase activity are frequently in endothelial cells of blood capillaries, mucous glandular cells (F3), microvilli of intestine (C6, CIO, D2, H21, P8, W7), bile canaliculi (D21, F27, W2), and placenta (W3), as well as in the brush border of the lumenal surface of epithelial cells of the proximal convoluted renal tubules (M22, Wl). The location of L-phenylalanine-sensitive alkaline phosphatase in human intestine and placenta is illustrated in Fig. 30. Electron micrographs (Fig. 31) show the details of the alkaline phosphatase, and illustrate the... 

Fig. 30. Enzyme staining reactions for L-phenylalanine-sensitive alkaline phosphatase in human placenta and intestine [conditions were those of Watanabe and Fishman (W7)l (a) human intestine in the presence of D-phenylalanine, X400 (b) high power view of human intestine showing brush border (arrow), terminal web, and apical concentration of alkaline phosphatase, xl200 (c) human intestine in the presence of L-phenylalanine, X400 (d) human placenta in the presence of D-phenylalanine, X400 (e) human placenta in the presence of L-phenylalanine, X400. Note that the enzyme location is on the peripheral absorptive surfaces of the intestine and placenta.

When rats are placed on a high-fat diet, the intestinal mucosa and the blood become enriched with alkaline phosphatase (F19, F20, Ml, M2). That the striated borders of the absorptive epithelial cells in rats possess the L-phenylalanine-sensitive alkaline phosphatase was proven by Watanabe and Fishman (W7). Human intestinal cells grown in tissue culture (W7) and human intestine exhibit L-phenylalanine sensitivity. Phosphatase has long been found to be present in feces (L8). 

More recently, studies in man have demonstrated the enrichment of the thoracic lymph with alkaline phosphatase during fat absorption (K6, K7). This phosphatase was L-phenylalanine-sensitive and was located as a slow-moving band on starch-gel electrophoresis, proving it to be intestinal in origin. 

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