Accumulation of phosphorus compounds in tissues and cultured skin fibroblasts in patients with hypophosphatasia

Patients with hypophosphatasia caused by a deficiency of alkaline phosphatase first showed marked accumulation of phosphoethanolamine and other phosphorus compounds in kidney and liver, while in placenta and intestine contents of these compounds were within a normal range. Furthermore, 32P-incorporation in cultured skin fibroblasts of patients with hypophosphatasia was increased about two to three times of control. FPLC chromatographic analysis also indicates that the accumulated phosphorus compounds in hypophosphatasia was smaller molecular phosphorus containing compounds. These data provide new pathophysiological aspect of hypophosphatasia.     

Pseudohypophosphatasia: aberrant localization and substrate specificity of alkaline phosphatase in cultured skin fibroblasts.

We explored the biochemical basis for the disorder pseudohypophosphatasia (PsHYPT) in one patient by examining the substrate specificity and localization of alkaline phosphatase (ALP) in cultured dermal fibroblasts. Despite substantial ALP activity, in cell homogenates, toward the artificial substrate 4-methyl-umbelliferyl phosphate (4-MUP), there was a marked deficiency in ALP activity toward the natural substrates pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), indicating altered substrate specificity. Furthermore, although 4-MUP phosphatase (4-MUP-P'tase) activity was predominantly localized as an ecto-enzyme, the small amount of PLP phosphatase (PLP-P'tase) activity was intracellular. This differential localization was apparent in intact cells, since (1) brief acidification of the medium at 4 degrees C inactivated a majority of the 4-MUP-P'tase activity but only 15% of the PLP-P'tase activity (in contrast to greater than 85% inactivation of both in homogenates), (2) greater than 70% of the 4-MUP-P'tase activity but only 30% of the PLP-P'tase activity was released by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion, and (3) degradation of extracellular PLP was less than 35% of that of disrupted cells. Both 4-MUP- and PLP-P'tase activities were predominantly in a lipid-anchored form that could be converted to a soluble, lipid-free form by PI-PLC digestion. Our findings suggest that the clinical and biochemical presentation of this PSHPT patient results from the production of two aberrant ALP species. One form of ALP has appropriate ectoorientation but is preferentially deficient in activity toward natural substrates; the other ALP species has appropriate substrate specificity but is sequestered from substrates by its intracellular location.     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Induction of alkaline phosphatase in cultured human fibroblasts. Comparison of normal cells and those from patients with cystic fibrosis.

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Characterization of a 5'-flanking region of the human liver/bone/kidney alkaline phosphatase gene: two kinds of mRNA from a single gene

We have cloned the human liver/bone/kidney alkaline phosphatase (ALPL) gene using a liver-type ALPL cDNA as a probe. The gene is divided into 12 exons, and is likely to exist as a single copy in haploid genome. As compared with the gene isolated using a bone-type ALPL cDNA (Weiss et al., J. Biol. Chem. 263, 12002-12010, 1988), another leader exon specific for the liver-type ALPL mRNA was assigned about 3.4 kb upstream from exon 2 and the alternative splicing in the first exon was indicated. RNA blot analysis showed that three species of mRNA of 2.5, 4.1 and 4.7 kilobases were detected in liver and developmentally regulated.     

The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism.

For the purified alkaline phosphatase from bone, the ability to catalyze a phosphate transfer reaction from p-nitrophenyl phosphate to two different hydroxy acceptor compounds, ethanolamine and glycerol, was established by identification of the formed phosphorylated products, phosphoethanolamine and glycerol 3-phosphate, respectively. In addition, a steady-state kinetic analysis of the hydrolysis of p-nitrophenyl phosphate in the presence of an added nucleophile, diethanolamine, gave rise to the proposal of a simple model for the kinetic mechanism of the enzyme. This mechanism includes a covalent phosphoryl enzyme intermediate, the dephosphorylation of which by water (k3) or a nucleophile (k4) is rate-determining. According to this model, in the presence of diethanolamine, k3 and k4 were determined to be 4.44 s-1 M-1 and 1000 s-1 M-1, respectively. Therefore, in vitro a suitable nucleophile, such as diethanolamine, seems to be a better phosphate acceptor than water. These results may suggest that alkaline phosphatase from bone could be well suited for catalyzing phosphate transfer reactions in vivo as well.     

Mucopolysaccharide accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV.

Increased concentrations of total sulfated mucopolysaccharides (MPS), threefold, and hyaluronic acid (HA), 10-fold, were found in ML IV fibroblast extracts when compared to normal controls. Such accumulations altered the distribution of MPS:HA comprised 70% of total MPS in ML IV but only 30% in control cells. Intracellular sulfated MPS was observed accumulating almost linearly in ML IV fibroblasts. "Pulse-chase" experiments indicate that both HA and the sulfated MPS remain in the ML IV cells for long periods of time; in control cells, they are rapidly removed as low molecular weight, dialyzable fragments. These data suggest that the MPS accumulation in ML IV fibroblasts, is the consequence of a catabolic block, probably involving the lysosome.     

Purification and characterization of alkaline phosphatase in cultured rat liver cells.

Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.     

Induction of alkaline phosphatase in cultured human fibroblasts: Comparison of normal cells and those from patients with cystic fibrosis

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP.Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

Cholesterol synthesis in cultured skin fibroblasts from patients with Huntington's disease

In view of the proposed membrane defect in Huntington's disease, cultured skin fibroblasts from healthy volunteers and patients with Huntington's disease were compared with respect to their ability to carry out de novo synthesis of cholesterol. At confluency, values for incorporation of [14C]acetate and 3H2O into cholesterol, and activities of HMG-CoA reductase (the rate-limiting enzyme in the cholesterol biosynthetic pathway), did not differ significantly in the Huntington's disease cells compared to the controls. Determinations of total cellular cholesterol gave similar ratios of cholesterol/protein and cholesterol/phospholipid in the Huntington's disease and control fibroblasts. The data suggest that the proposed generalized cell membrane abnormality in Huntington's disease cannot be attributed to a defect in the cholesterol biosynthetic pathway.     

Properties of beta-glucosidase in cultured skin fibroblasts from controls and patients with Gaucher disease.

Membrane-bound beta-glucosidase from cultured skin fibroblasts can be solubilized in an active form by treatment of membrane preparations with a mixture of Triton X-100 and sodium taurocholate. Several properties of the solubilized enzyme have been studied in fibroblasts from normal, healthy individuals and from 14 patients with different clinical forms of Gaucher disease. The patients studied were classified as follows: group 1 consisted of 10 chronic patients, all (with one exception) of Ashkenazi Jewish origin; group 2 consisted of three black American patients with severe visceral symptoms, manifest from early childhood, but with no apparent neurological involvement; and group 3 consisted of a single white patient with the classical infantile form of the disease. Specific beta-glucosidase activity ranged from 6.6% to 16.5% mean control value in group 1 patients and from 4.1% to 5.8% in groups 2 and 3. When compared with the enzyme from control fibroblasts, the enzyme from chronic Gaucher patients (group 1) was more rapidly inactivated at 50 degrees C, had an altered pH curve, was less effectively inhibited by deoxycorticosterone-beta-glucoside, and was more effectively inhibited by deoxycorticosterone. The enzyme from patients in groups 2 and 3 was qualitatively indistinguishable from the control enzyme in terms of these parameters. No differences in Km (4-methylumbelliferyl-beta-glucoside) or sedimentation coefficient were found between the beta-glucosidases from control and Gaucher cells. The results demonstrate that cells from Ashkenazi Jewish patients with the chronic form of Gaucher disease contain a structurally altered form of beta-glucosidase. This enzyme differs both from normal beta-glucosidase and from the residual enzyme in patients of different ethnic origin and with clinically more severe forms of the disease.     

Hyaluronate synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate secreted into the medium by Werner's fibroblasts was 2-3-times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibroblasts were then cultured in the presence of [3H]glucosamine, and the amount of [3H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain length of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in the matrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.