Alkaline phosphatase human placental

Alkaline phosphatase, human placental Whole plant, hydroponic Nicotiana tabacum (tobacco) A. tumefaciens transformation of leaf explant Mannopine synthase (mas2 ) N ot reported 20 Jig day"1 g"1 root dry weight (e) 3 % of total medium protein (e) 72... 

Jemmerson, R., and Agre, M. (1987) Monoclonal antibodies to different epitopes on a cellsurface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold. J. Histochem. Cytochem. 35, 1277-1284. 

Serum alkaline phosphatase elevations have been reported following administration of salt-poor albumin (B5). Placenta is very rich in a heat-stable alkaline phosphatase, and albumin prepared from placental blood has a high activity of this enzyme. In one cirrhotic patient who received 1-6 units per day of albumin obtained from pooled human blood and/or human placenta, the alkaline phosphatase before infusion was 5 Bodansky units and by the thirteenth day of administration had reached a value of 160 units. The physician administering the albumin at first thought the patient was having a severe toxic liver reaction and stopped the therapy. The alkaline phosphatase then started to go down and within 10 days returned to normal levels. Analysis of the albumin indicated that it contained 470 units of alkaline phosphatase activity and was probably responsible for the observed elevations in the serum enzyme activity. Albumin prepared from venous blood did not cause an alkaline phosphatase elevation, but placenta-albumin caused elevations with a half-life of about 8 days (Ml). 

Amino Acid Composition of Human Placental Alkaline Phosphatase... 

Human Placental Alkaline Phosphatase Relative Reaction Rates and Michaelis Constants for Various Substrates0... 

Makiya, R. and T. Stigbrand. 1992. Placental alkaline phosphatase is related to human IgG internalization in FIEp2 cells. Biochem. Biophys. Res. Commun. 182 624-630. 

COS cells have been used for the production of recombinant proteins, including HSV-1 glycoprotein B, ricin B chain, placental alkaline phosphatase, thrombomodulin, CD7, von Willebrand factor, human dihydropteridine reductase, human P-glucuronidase, interleukin 5 and human interferon-... 

A degree of organ specificity for substrate is to be expected. Thus, a substrate preference known is the ability of intestinal alkaline phosphatase to hydrolyze o-carboxyphenylphosphate more rapidly than other substrates (Fll, F13). Further, placental alkaline phosphatase is stated to hydrolyze p-nitrophenyl phosphate less rapidly than j8-glycerophos-phate (SI). Although ethanolamine phosphate is hydrolyzed preferentially by rat liver alkaline phosphatase, such a preference was missing from human liver preparations (Fll). 

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. 

These are listed in Table 7. Just as in the case of substrate, organ-specific pH optima have been observed. Thus, for example, purified human placental alkaline phosphatase exhibits an optimum pH of 10.6 in contrast to 9.8 for the human intestinal preparation (Fig. 7). 

Fig. 8. Relationship between pX versus pH for intestine (rat) and placental (human) alkaline phosphatases. Filled circles represent intestine and open triangles human placenta. The plot for intestinal enzyme is made according to Ghosh and Fishman (G5). Ammonium sulfate fractionation was omitted during the purification of the placental enzyme.

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). 

Fig. 17. Starch-gel patterns of Sephadex G-200 gel filtrates of human placental alkaline phosphatase showing the fractionation of variants A and B. The fraction numbers are indicated. The direction of migration was from bottom to top (anode).

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

The resolution of the isozymes of human alkaline phosphatase in normal individuals by starch-gel electrophoresis was systematically studied in 1961 by Boyer, who observed a characteristic alkaline phosphatase pattern similar in pregnancy sera and in placenta (B38). With regard to placenta, recent work (H5, R15, R16) has indicated genetic variation of placental alkaline phosphatase in human placenta when the starch-gel electrophoresis is carried out at two different pH s (8.6 and 6.0). Other tissues could not be differentiated by their starch-gel patterns by Boyer (B38, B39). 

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. 29. Optimum pH of hydrolysis of human placental alkaline phosphatase by neuraminidase. The liberated sialic acid was measured by the Warren-thiobarbiturate procedure, using iV-acetylneuraminic acid as the standard (G6a).

Harris and his group (R15, R16) in London recently initiated genetic studies on human placental alkaline phosphatase, and demonstrated phenotypic differences in this enzyme. In this study, butanol extracts of alkaline phosphatase from each of 338 placentas were prepared, and the enzyme preparations were subjected to starch-gel electrophoresis at two different pH s (8.6 and 6.0). The electrophoretic patterns obtained were classified in six different groups representing six distinct phenotypes. The... 

This order of evaluation (biochemical, first starch gel, second) is amply justified from our own experiences and the consensus of opinion at a recent symposium on multiple molecular forms of enzymes. In particular, heterogeneity of human intestinal alkaline phosphatase on starch gel was reported by Moss (M34) and Fishman and Kreisher (F9, K25). A certain amount of LPSAP intestinal enzyme may occupy the same position that liver alkaline phosphatase migrates to. On the other hand, more than one band in nonliver positions can be produced by intestine. Another similar situation is seen with placental alkaline phosphatase, which may show three bands. Consequently, the starch-gel data can be correlated with biochemical studies only if the nature and organ source of the preparation are known in advance. 

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). 

Boyer (B38) was the first to indicate the polymorphism of human placental alkaline phosphatase by starch-gel electrophoresis. He suggested also the existence of genetic variants of the placental isoenzymes, which were investigated in greater detail by Harris and his collaborators (R16, R16). 

G6. Ghosh, N. K., and Fishman, W. H., Characterization of human placental alkaline phosphatase isoenzymes. Federation Proc. 26, 558 (1967). 

R16. Robson, E. B., and Harris, H., Further studies on the genetics of placental alkaline phosphatase. Ann. Human Genet. (London) 30, 219-232 (1966). 

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