Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. We used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. We observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolishes the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.

IntroductionHypophosphatasia features selective deficiency of activity of the tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (ALP) To test whether human PALP and TNSALP are physiologically active toward the same substrates, we studied PEA, PPi, and PLP levels during and after pregnancy in three women who are carriers for hypophosphatasia. Hypophosphatasemia corrected during the third trimester because of PALP in maternal blood. Blood or urine concentrations of PEA, PPi, and PLP diminished substantially during that time. After childbirth, maternal circulating levels of PALP decreased, and PEA, PPi, and PLP levels abruptly increased. In serum, unremarkable concentrations of IALP and low levels of TNSALP did not change during the study period.We conclude that PALP, like TNSALP, is physiologically active toward PEA, PPi, and PLP in humans. We speculate from molecular/crystallographic information, indicating significant similarity of structure of the substrate-binding site of ALPs throughout nature, that all ALP isoenzymes recognize these same three phosphocompound substrates.

1. Inhibition studies have been carried out on a series of ALPs derived from liver, bone, kidney, placenta and intestine, using L-phenylalanine, L-homoarginine, L-leucine, L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine as inhibitors. 2. No differences between liver, bone and kidney ALPs with any of the inhibitors were observed. 3. L-phenylalanine and L-homoarginine give a major degree of discrimination between liver/bone/kidney ALP on the one hand, and placental and intestinal ALPs on the other. L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine give a major degree of discrimination between placental ALP on the one hand, and intestinal ALP and liver/bone/kidney ALP on the other. L-leucine discriminates between the three classes, but to a lesser degree. Minor degrees of discrimination between placental and intestinal ALPs occur with L-phenylalanine and L-homoarginine and between intestinal and liver/bone/kidney ALPs with L-leucyl-glycyl-glycine and L-phenylalanyl-glycyl-glycine. By using an appropriate combination of inhibitors the ALPs can be separated into three clearly distinct categories: placental, intestinal and liver/bone/kidney. 4. The six common placental ALP phenotypes as defined by electrophoresis show identical inhibition profiles with the series of inhibitors. The same profile was found for several rare electrophoretic variants. However, two rare electrophoretic variants (P-187 and P-92) each encountered once in a sample of 225 plancentae, showed significantly deviant inhibitions with the various inhibitors and also differed from each other. From the electrophoretic patterns, both of these rare phenotypes appear to be heterozygotes. P-187 probably corresponds to the so-called D-variant previously described. P-92 represents a new type of placental ALP variant with an aberrant inhibition profile. In both cases the particular rare allele concerned evidently alters the primary structure of the enzyme so that it has an altered electrophoretic mobility and also an altered sensitivity to inhibition for each of the different inhibitors. 5. Treatment of the various ALPs with neuraminidase to remove sialic acid residues does not affect their inhibition characteristics or activities.

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Starch gel electrophoresis and inhibition studies with L-phenylalanine, L-homoarginine, L-leucine, Lleucylglycylglycine, and L-phenylalanylglycylgylcine were carried out on a series of human-alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] derived from fetal and adult liver, kidney, bone, and intestine. No differences between adult and fetal liver, kidney, or bone alkaline phosphatases were observed by either electrophoretic or inhibition studies. However, the fetal intestinal enzyme could be clearly distinguished from the adult intestinal enzyme by its greater anodal electrophoretic mobility and its retardation after treatment with neuraminidase. Even after extensive neuraminidase treatment, its anodal mobility was still slightly greater than that of adult intestinal alkaline phosphatase. Fetal and adult intestinal enzymes showed the same inhibition profiles with the series of inhibitors both before and after treatment with neuraminidase.