L-Phenylalanine-sensitive alkaline phosphatase

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... 

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. 

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... 

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). 

A systematic study with modern methods has been undertaken by Lincheer et al. (L14) of the relationship of intestinal alkaline phosphatase and glucose absorption. Apparently glucose is capable of stimulating the intestinal mucosa to secrete an L-phenylalanine-sensitive alkaline phosphatase. 

The identity of the slow-moving band observed in all of these studies is the intestine, based on its L-phenylalanine sensitivity (F9, K25, R7, S49, N20, W6) and resistance to neuraminidase (R7). This is even more certain following the demonstration of an elevation in thoracic lymph and in blood serum of L-phenylalanine-sensitive alkaline phosphatase in subjects absorbing fat. The slow-moving band becomes intensified in sera of individuals who are secretors (K7, L2). 

Stolbach, L. L., Krant, M. J., Inglis, N. I., and Fishman, W. H., Correlation of serum L-phenylalanine sensitive alkaline phosphatase, derived from intestine, with the ABO blood group of cirrhotics. Gastroenterology 103, 819-827 (1967). 

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. 

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. 

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

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... 

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. 

Since there is no difference in the amount of alkaline phosphatase in the intestinal mucosa of subjects of various blood types and secretor status (L2) and since this enzyme is presumed to be L-phenylalanine-sensitive (L14), then the difference in the relative amount of heat-stable LPSAP in persons with or without the slow band may relate to events... 

With the aid of photomicrographs of the L-phenylalanine-sensitive placental alkaline phosphatase, one can picture the placental villi in contact with the maternal circulation (H3) and so enriching it with alkaline phosphatase. In support of this view, we have demonstrated a progressive rise in pregnancy of heat-stable LPSAP with an optimum pH of 10.7. 

F8. Fishman, W. H., and Green, S., Automated differential isoenzyme analysis. I. L-phenylalanine-sensitive isoenzymes of human serum alkaline phosphatase. Enzymologia, 38, 89-99 (1967). 

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). 

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

We assume that normal individuals have the same organ sources of serum alkaline phosphatase whether or not the slow intestinal band is present. Thus, persons with the band have more total and heat-stable LPSAP and those lacking the band, more heat-stable non-LPSAP. This can be explained if some of the intestinal alkaline phosphatase, before, during, or after absorption loses its sensitivity to L-phenylalanine but... 

The alkaline phosphatase of both human intestine and placenta are L-phenyl-alanine-sensitive and undergo uncompetitive inhibition to the same extent (nearly 80%) by 0.005 M L-phenylalanine. However, we have been able to find several distinguishing biochemical characteristics of the two enzymes (1) the anodic mobility of intestinal alkaline phosphatase remains unchanged after neuraminidase treatment, whereas the placental enzyme is sialidase-seusitive and hence the electrophoretic mobility on starch gel is considerably reduced by such treatment, (2) the Michaelis constant of placental alkaline phosphatase at a definite pH is appreciably higher than that of the intestinal enzyme (at pH 9.3 the Km values of placenta and intestine are 316 and 160 ixM, respectively), and (3) the pH optima (with 0.018 Af phenyl phosphate as substrate) of the two enzymes are different the values for intestinal and placental enzymes with 0.006 Af n-phenylalanine are 9.9 and 10.6, respectively, and the respective values in the presence of 0.005 Af L-phenylalanine are 10.2 and 11.1. Finally, contrary to the behavior of intestinal alkaline phosphatase, placental enzyme is completely heat stable (P19). 

The portal blood-borne alkaline phosphatase from the intestine undergoes mixing in the liver with the enzyme that has traveled the lymphatic route. The proportions of these two populations of enzyme molecules, and their heat lability and sensitivity to L-phenylalanine are under the control of a number of physiological variables already discussed. 

In brush border of intestine also a relatively anion-insensitive ATPase could be found [37]. Previous negative findings [50] could be explained by the fact that the chelator EDTA, which inactivates this enzyme [51], was present in the incubation medium. This inactivation can be overcome by addition of excess [51]. This finding and the fact that the anion-sensitive ATPase in intestinal brush-border membranes can be inhibited by L-phenylalanine and L-cysteine suggests that the alkaline phosphatase activity and the anion-sensitive ATPase activity originates from a single enzyme [37,41]. The same conclusion has been reached for the anion-sensitive ATPase of brush border from human placenta [41], but not for the enzyme from rat kidney [16], where the alkaline phosphatase activity is inhibited by l-p-bromo-tetramisole, whereas the anion-sensitive ATPase activity is not affected. 

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