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Showing papers on "Alkaline phosphatase published in 1978"


Journal ArticleDOI
TL;DR: It was found that the unlabelled antibody immunohistological procedure can be shortened without loss of sensitivity by carrying out two of the incubation steps simultaneously, and the total duration of this double immunostaining procedure is only a few minutes longer than that required for detection of a single antigen.
Abstract: The use of alkaline phosphatase in an immunoenzymatic procedure for the localisation of antigens in paraffin sections or cell smears is described. The results of this method, when applied to the detection of immunoglobulins, lysozyme, or lactoferrin, were comparable in intensity and clarity to those obtained with the PAP immunoperoxidase procedure. Furthermore, double immunoenzymatic labelling (with alkaline phosphatase and peroxidase) of two cellular constituents in a tissue section is possible, the brown peroxidase reaction product contrasting well with the blue alkaline phosphatase product. Since the two antibody 'sandwiches' are applied simultaneously rather than sequentially the total duration of this double immunostaining procedure is only a few minutes longer than that required for detection of a single antigen. It was also found that the unlabelled antibody immunohistological procedure (whether used in conjunction with alkaline phosphatase or peroxidase) can be shortened without loss of sensitivity by carrying out two of the incubation steps simultaneously.

394 citations


Journal ArticleDOI
TL;DR: It is concluded that 1alpha,25(OH)2D3 is not the sole biologically active metabolite of vitamin D in man and that the subsequent rise in serum phosphate concentrations along with the direct actions of 1 alpha-25-hydroxyvitamin D3, 24,25 (OH) 2D3, and possibly 25(OH-D3 on bone cells all participate in the restoration of normal bone formation and bone mineralization in vitamin D-deficient man.
Abstract: A comparison was made of the biochemical and osseous effects of 25–hydroxyvitamin D3[25(OH)D3], lα–25–hydroxyvitamin D3[lα,25(OH)2D3], and 24,25–dihydroxyvitamin D., [24,25(OH)2D3] in adult vitamin D-deficient man. Administration of 50 jug/d of 25(OH)D3 for 8 weeks led to a return of the mineralization front to normal associated with a return of Tmpo4/GFR to normal, an increase in serum phosphate and calcium concentrations, a fall in serum IPTH, and a rise in serum alkaline phosphatase activity. Giving 2.5 jug/d of la,25(OH)2D3 did not produce these effects. Administration of lα,25(OH)2D3 caused an increase in intestinal calcium absorption, and a rise in serum calcium associated with a fall in serum immunoreactive parathormone (IPTH) concentrations but no sustained rise in either alkaline phosphatase, serum phosphate concentration, nor in Tmpo4/GFR. Its administration caused an increase in the extent of the osteoclastic bone resorption surface but the extent of the mineralization front remained subnormal....

195 citations


Journal ArticleDOI
TL;DR: It was concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.

173 citations


Journal ArticleDOI
TL;DR: Endocytosis of ;low-uptake' forms of alpha-N-acetylglucosaminidase and alpha-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that low-uptak' forms are either contaminated with ;high-uptakes' forms or are internalized via the same route as ; high-uptaking' forms.
Abstract: Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.

165 citations


Journal ArticleDOI
TL;DR: Findings indicate that Na+,K+-ATPase is localized to the sinusoidal and lateral portions of the rat hepatocyte plasma membrane and is not detectable on the bile canaliculus where alkaline phosphatase is confined.
Abstract: The enzyme Na+,5+-ATPase was cytochemically localized in the rat hepatocyte by a modification of the Ernst potassium-dependent nitrophenyl phosphatase technique. Measurement of nitrophenol release from 50-micrometer liver slices confirmed the presence of ouabain-inhibitable nitrophenyl phosphatase activity that increased over the 30-min incubation period. Electron micrographs demonstrated that sinusoidal and lateral membrane reaction product deposition was K+-dependent, Mg++-dependent, inhibited by ouabain but not by alkaline phosphatase inhibitors, and was localized to the cytoplasmic side of the membrane. In contrast, canalicular reaction product was K+-independent, Mg++-dependent, inhibited by alkaline phosphatase inhibitors but not by ouabain, and was localized to the luminal side of the membrane. These findings indicate that Na+,K+-ATPase is localized to the sinusoidal and lateral portions of the rat hepatocyte plasma membrane and is not detectable on the bile canaliculus where alkaline phosphatase is confined. This basolateral localization of Na+,K+-ATPase is similar to that found in epithelia where secretion is also directed across the apical membrane.

156 citations


Journal ArticleDOI
TL;DR: 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 and L‐leucyl‐ glyoyl‐glycine as inhibitors.
Abstract: 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.

140 citations


Journal ArticleDOI
01 Jan 1978
TL;DR: By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.
Abstract: Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.

140 citations


Journal ArticleDOI
TL;DR: The ability of either inhibitor to block 1,25(OH)2D3-mediated calcium transport despite inhibition of CaBP production and alkaline phosphatase activity (by cycloheximide) indicates that de novo RNA and protein synthesis, and in particular CaBP and alkalinase, are not required for the 1, 25(OH), 2D3 stimulation of calcium transport.

132 citations


Journal ArticleDOI
TL;DR: Enzyme activity was optimal in the presence of 10−5 m orthomosphate but was markedly depressed at concentrations above 10−4m, and the possible implications of these results with respect to mechanisms involved in the assimilation of phosphorus in the VA mycorrhizal system and/or the establishment of VA my Corrhiza-specific phosphatase are discussed.
Abstract: Soluble alkaline phosphatase specific to vesicular-arbuscular mycorrhizal infection has been demonstrated in enzyme extracts from onion roots inoculated with Glomus mosseae. A close correlation existed between the mycorrhiza-specific phosphatase activity and development of both the infection and the host plant. Maximum activity occurred whilst the infection was still young (100% arbuscular) and coincided with the start of the mycorrhizal growth response, declining afterwards as plant development and infection continued. Enzyme activity was optimal in the presence of 10−5 m orthomosphate but was markedly depressed at concentrations above 10−4m. The properties of mycorrhiza-specific alkaline phosphatase were characteristic of alkaline phosphomonesterase (EC 3.1.3.1.): optimal activity at alkaline pH; inhibition by metal chelating agents, certain cations and orthophosphate; requirement for Mg2+ and Mn2+ ions; hydrolysis of β-glycerol, phenyl and naphthyl phosphates; inability to hydrolyse more complex phosphate esters. The possible implications of these results with respect to mechanisms involved in the assimilation of phosphorus in the VA mycorrhizal system and/or the establishment of VA mycorrhizal infection are discussed.

108 citations


Journal ArticleDOI
TL;DR: The morphology and histochemistry of dissociated newborn rat brain was studied in tissue culture using direct microscopy of developing cells, electron microscopy and the alkaline phosphatase activity to identify the capillary endothelial cells.
Abstract: The morphology and histochemistry of dissociated newborn rat brain was studied in tissue culture. Direct microscopy of developing cells, electron microscopy and the alkaline phosphatase activity were used to identify the capillary endothelial cells.

95 citations


Journal ArticleDOI
TL;DR: A survey of intestinal samples from fetuses and premature infants of various gestational ages indicated that the changeover from the synthesis of fetal to adult intestinal enzyme begins at about 28-32 weeks of gestation.
Abstract: Starch gel electrophoresis and inhibition studies with L-phenylalanine, L-homoarginine, L-leucine, L-leucylglycylglycine, and L-phenylalanylglycylglycine 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. A survey of intestinal samples from fetuses and premature infants of various gestational ages indicated that the changeover from the synthesis of fetal to adult intestinal enzyme begins at about 28-32 weeks of gestation. The difference between the fetal and adult forms of intestinal alkaline phosphatase may represent the expression of different gene loci or a difference in post-translational modification.

Journal ArticleDOI
TL;DR: Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.
Abstract: Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine. Agarose coupled to L-histidyldiazobenzylphosphonic acid was found to be a highly effective adsorbent. In order to understand the large differences in binding capacity observed with derivatized agaroses, inhibition of alkaline phosphatase by phosphonic acid ligands, and related phosphonic acids, was measured. The results of affinity chromatography and inhibition studies were in good agreement, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.

Journal ArticleDOI
TL;DR: It is concluded that the chick cell-derived oligonucleotide inhibitor has a structure that is closely related or identical to that of the inhibitor made in the mouse system, and that both preparations inhibit cell-free protein synthesis in a non-species-specific manner.
Abstract: Cytoplasmic extracts of interferon-treated primary chick embryo cells contain an enzyme activity that synthesized an inhibitor of chick cell-free protein synthesis. The same activity was detected in extracts of cells treated with mock preparations of interferon, but at <0.3% of the level found in interferon-treated cell extracts. The enzyme was activated by double-stranded RNA and could be isolated by binding to columns of poly(I)-poly(C)-agarose. In the column-bound state, the enzyme reacted with ATP to synthesize the inhibitor, which could then be continuously eluted from the column. The inhibitor was purified and its structure and function were compared with those of the low molecular weight inhibitor of protein synthesis made by an enzyme from interferon-treated mouse L cells. The avian and mammalian inhibitors comigrated on thin layers of polyethyleneimine-cellulose during chromatography in three different solvent systems, and they coeluted as a series of peaks from columns of DEAE-cellulose during sodium chloride gradient elution. Digestion with bacterial alkaline phosphatase or snake venom phosphodiesterase yielded products that similarly comigrated. Functionally, the two inhibitors were interchangeable: both inhibited protein synthesis in extracts of mammalian and avian cells, producing 50% inhibition at a concentration of about 0.3 nM (AMP equivalents). We conclude that the chick cell-derived oligonucleotide inhibitor has a structure that is closely related or identical to that of the inhibitor made in the mouse system, and that both preparations inhibit cell-free protein synthesis in a non-species-specific manner.

Journal ArticleDOI
TL;DR: The effects of glycosidase digestion on the alkaline phosphatase isoenzymes, changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated.

Journal ArticleDOI
TL;DR: The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol.

Journal ArticleDOI
TL;DR: The hypothesis that the deleterious effect of ethanol oxidation on pyridoxal phosphate metabolism is mediated at least in part by acetaldehyde which displaces this coenzyme from protein binding, thereby enhancing its degradation is supported.
Abstract: Previous studies in vivo and with isolated perfused rat livers have suggested that the deleterious effect of ethanol on hepatic pyridoxal 5'-phosphate metabolism is mediated by acetaldehyde. Inasmuch as acetaldehyde has no effect on the synthesis of pyridoxal phosphate, it has also been postulated that acetaldehyde accelerates pyridoxal phosphate degradation by displacing this coenzyme from binding proteins, which protect it against hydrolysis. To test these hypotheses, studies have been performed with isolated rat hepatocytes, subcellular fractions of rat liver, and human erythrocytes. Ethanol oxidation lowered the pyridoxal phosphate content of isolated liver cells when acetaldehyde oxidation was inhibited by either disulfiram or prior treatment of rats with cyanamide. Additions of 7.5 mM acetaldehyde alone at 40-min intervals to cell suspensions decreased hepatic pyridoxal phosphate content only slightly because acetaldehyde was rapidly metabolized. However, when acetaldehyde oxidation and reduction were inhibited by cyanamide treatment and by 4-methyl-pyrazole and isobutyramide, respectively, a 40% decrease in hepatic pyridoxal phosphate content was observed in 80 min of incubation. In equilibrium dialysis experiments, acetaldehyde, 7.5 and 15 mM, displaced protein-bound pyridoxal phosphate in undialyzed hepatic cytosol and in hemolysate supernate containing added pyridoxal phosphate. In the presence of alkaline phosphatase, acetaldehyde accelerated the degradation of pyridoxal phosphate in dialyzed hemolysate supernate and hepatic cytosol with added pyridoxal phosphate. Acetaldehyde also inhibits tyrosine aminotransferase. The kinetics of inhibition were mixed competitive-noncompetitive with respect to pyridoxal phosphate. These observations support the hypothesis that the deleterious effect of ethanol oxidation on pyridoxal phosphate metabolism is mediated at least in part by acetaldehyde which displaces this coenzyme from protein binding, thereby enhancing its degradation.

Journal ArticleDOI
TL;DR: Evidence is presented to indicate differences in the leucylpeptide backbones of the antibiotic-sensitive cells and the drug-resistant DC-3F/AD X cells as revealed by sodium dodecyl gel electrophoresis.
Abstract: Plasma membrane proteins and glycoproteins have been isolated from Chinese hamster cells of the spontaneously transformed DC-3F parental cell line and the DC-3F/AD X line with a high level of acquired resistance to actinomycin D Plasma membrane preparations from both cell lines band at 116 g/ml after isopycnic centrifugation We present evidence to indicate differences in the leucylpeptide backbones of the antibiotic-sensitive cells and the drug-resistant DC-3F/AD X cells In addition, there are differences in the plasma membrane glycopeptides of the two cell lines as revealed by sodium dodecyl gel electrophoresis Drug-resistant cells synthesize a surface glycopeptide which is much larger than the major one present on the drug-sensitive cells Both of these cell lines are devoid of 5′-nucleotidase and alkaline phosphatase activities The role of plasma membrane protein differences in drug-resistant cells is discussed

Journal ArticleDOI
TL;DR: In strips of duodenum from 14-day chick embryos explanted into defined medium, alkaline phosphatase (ALP) activity accumulated in the tissue at a faster rate than in vivo for about 48 hr, but failed to increase thereafter.

Journal ArticleDOI
01 Jun 1978-BJUI
TL;DR: Bone scanning with 99mTc-Sn-HEDP, radiographic skeletal survey and determination of plasma acid and alkaline phosphatase values were carried out in a consecutive series of 90 untreated patients with carcinoma of the prostate to support the validity of scan positive--X-ray negative findings.
Abstract: Summary— Bone scanning with 99mTc-Sn-HEDP, radiographic skeletal survey and determination of plasma acid and alkaline phosphatase values were carried out in a consecutive series of 90 untreated patients with carcinoma of the prostate. 99mTc-Sn-HEDP provided satisfactory bone imaging and was convenient in use. The addition of bone scanning to radiographic survey increases the detection rate of skeletal metatases by 16%. Radiography increases the accuracy of bone scanning by identifying false positive scans due to benign disease and false negative scans when there are diffuse symmetrical bony metastases. The plasma phosphatases alone are less accurate staging tests. The acid phosphatase data support the validity of scan positive—X-ray negative findings. Bone scan abnormalities due to secondary deposits usually precede elevation of plasma alkaline phosphatase.

Journal ArticleDOI
TL;DR: It has been shown that the detergent and papain forms of alkaline phophatase are dimers consisting of two apparently identical subunits whose molecular weights are 64 000 and 61 000, respectively.

Journal ArticleDOI
TL;DR: It is suggested that phytase activity is a manifestation of alkaline phosphatase and the significance of this in relation to phytate-induced Zn deficiency is discussed.
Abstract: 1. The activities of alkaline phosphatase (EC 3.1.3.1) and phytase (EC 3.1.3.8) were similarly distributed in the small intestine of rats. Regional differences in activity were reflected by similar differences in the capacity of ligated intestinal segments to hydrolyse phytate in vivo. Activities were greatest in the duodenum and lowest in the terminal ileum. 2. Specific activities of both enzymes were tenfold greater in the brush border fraction of duodenal mucosa compared with entire mucosal homogenates. 3. Brush-border alkaline phosphatase and phytase activities required both magnesium and zinc ions for maximal activity. 4. Zn deficiency induced by feeding a diet low in Zn (0.5 mg Zn/kg) caused similar reductions in activity of both enzymes. 5. Zn deficiency induced by feeding diets marginally adequate in Zn (12 mg/kg) and phytate (10 g/kg) caused reductions in alkaline phosphatase, phytase activities and phytate hydrolysis in vivo. 6. It is suggested that phytase activity is a manifestation of alkaline phosphatase and the significance of this in relation to phytate-induced in Zn deficiency is discussed.

Journal ArticleDOI
TL;DR: The hypothesis that the growth of mammalian cells is regulated by hormones is supported by considerable evidence and two rat pituitary cell lines, GH3 and GC, and a human cervical carcinoma cell, HeLa S-3, have been grown indefinitely in serum-free (SF) hormone-supplemented medium.
Abstract: The hypothesis that the growth of mammalian cells is regulated by hormones is now supported by considerable evidence. Two rat pituitary cell lines, GH3 and GC, a mouse melanoma, M2R (B16), and a human cervical carcinoma cell, HeLa S-3, have been grown indefinitely in serum-free (SF) hormone-supplemented medium. No visible changes of growth characteristics were observed in the cells grown continuously in the SF condition. However, changes in the activity of a plasma membrane enzyme, alkaline phosphatase, and in the relative intensity of surface proteins that are labeled by the [125I] lactoperoxidase technique were found in HeLa cells grown in the SF condition. To study the role of hormones required in the regulation of cell growth, HeLa cells were grown in the absence of one of the required hormones. The following results were obtained. Epidermal growth factor is probably involved in the regulation of the synthesis of macromolecules such as RNA and of the protein content per cell. Transferrin, the accessory factor in the SF condition, supplies iron for cells. The two basic peptides in this SF system, fibroblast growth factor and insulin, are probably involved in the balance of nutrients and energy inside the cell. The replacement of F12 medium with a better-balanced medium, MCDB 105, can mimic the requirements for these two peptides. The steroid hydrocortisone (HC) is probably involved in alteration of the cell surface. This is indicated by the effects of HC on cell morphology, rate of detachment from the dish, and the pattern of [125I] lactoperoxidase labeling of surface proteins. In addition, it is necessary to change the medium more frequently to maintain the culture in the medium without HC. This observation suggests that HC may be involved in the control of homeostatic properties of the cell surface. The production of rat prolactin by GH3 cells was also studied. GH3 cells in the SF condition produce 1.6 microgram prolactin per 10(5) cells in 24 h, while 2.4 microgram is produced in the presence of serum. Prolactin production in the SF condition is enhanced by the presence of thyrotropin-releasing hormone and inhibited by triiodothyronine (T3). T3 is the major growth factor for these cells. Without it cell growth is severely limited, while prolactin production is elevated. This result suggests that the GH3 cell line in the SF condition may be an ideal system for the study of hormonal regulation of cell growth and specific gene expression.

Journal ArticleDOI
TL;DR: There was some evidence from the short-term studies that calcitonin produced a more rapid fall in skeletal blood flow than in alkaline phosphatase, and Glomerular filtration rate appeared to increase transiently in response to calciton in patients re-studied.
Abstract: 1. Blood flow to the skeleton was measured by the 18F clearance method of Wooton, Reeve & Veall (1976) in 24 patients with untreated Paget's disease. In every patient but one, resting skeletal blood flow was increased. There was a significant positive correlation between skeletal blood flow and serum alkaline phosphatase and between skeletal blood flow and urinary total hydroxyproline excretion. 2. Fourteen patients were re-studied after they had received short-term (7 days or less) or long-term (7 weeks or more) calcitonin. Skeletal blood flow, alkaline phosphatase and urinary hydroxy-proline excretion fell towards normal in every case. There was some evidence from the short-term studies that calcitonin produced a more rapid fall in skeletal blood flow than in alkaline phosphatase. 3. Glomerular filtration rate appeared to increase transiently in response to calcitonin.

Journal ArticleDOI
TL;DR: The isolation and purification of forphenicine was reported on, which inhibits alkaline phosphatase and enhanced delayed-type hypersensitivity and increased the number of antibodyforming cells.
Abstract: Sir: Enzyme inhibitors can be powerful tools in analyzing the various aspects of homeostasis in living organisms and even in understanding disease processes. As we reported, small molecular inhibitors of endopeptidases, exopeptidases and glycosidases have been found in culture filtrates of actinomycetes\"-4'. Recently we demonstrated that activities of aminopeptidases, alkaline phosphatase and other enzymes are located on the surface of various types mammalian cells\"\" and that these enzymes are indispensable in various functions of cells''81. As reported previously, our experiments with bestatins'10', a specific inhibitor against aminopeptidase B and leucine aminopeptidase, demonstrated that it bound to FM3A and mouse spleen lymphocyte cells\"'. Bestatin enhanced delayedtype hypersensitivity and it produced immune resistance to experimental animal tumors\"\"'. We, therefore, searched for an inhibitor of alkaline phosphatase and found a new compound named forphenicine. As reported in another paper, forphenicine enhanced delayed-type hypersensitivity and increased the number of antibodyforming cells\". In this communication, we will report on the isolation and purification of forphenicine which inhibits alkaline phosphatase. The method described by BESSEY121 for determination of alkaline phosphatase (EC 3.1.3.1.) activity was modified for the quantitative assay of its inhibitors as follows\"': to 0.02 ml of 0.1 M p-nitrophenyl phosphate (Daiichi Pure Chem. Co., Tokyo) in distilled water, 0.06 ml of 0.1 M carbonate buffer at pH 9.0, 0.01 ml of 0.2 M of magnesium chloride and 0.1 ml of distilled water with or without a test material were added; after 3 minutes at 37°C, 0.01 ml (0.25 ug of alkaline phosphatase of chicken intestine, Nutritional Biochemicals Co., U.S.A.) was added and the reaction mixture was incubated for 20 minutes at 37°C; after the incubation, the reaction was stopped by addition of 1.5 ml of 0.15 N NaOH and the extinction of the supernatant of the centrifuged solution was read at 400 nm. The reaction was also carried out without addition of enzyme solution and the result was taken as blank. The concentration of forphenicine required for 50% inhibition (IC,,) was calculated as described previously\"'. We observed that many strains of various species of actinomycetes produce an agent active in inhibiting alkaline phosphatase, and we isolated an active agent from the strain MC974-A5 which was isolated from the soil sample collected in Ito City, Shizuoka prefecture and was classified as Actinomyces fulvoviridis var. acarbodicus. Forphenicine was produced by shaking culture or tank fermentations of the strain MC974-A5 in a medium containing soybean meal 1.5%, NaCI 0.3 %, MgSO4.7H,O 0.1 %, KZHPO4 0.1 %, CuS04. 5H2O 0.0007 %, FeSO4.7 H20 0.0001 %, MnCl2.4H,O 0.0008%, ZnSO4.7H2O 0.0002 (pH was adjusted to 7.2 with 2 N NaOH before sterilization). The maximum production of forphenicine was attained on the 4 5th day of the shaking culture or on the 4th day of tank fermentation and maintained for 2 3 days thereafter. Extraction and purification processes are shown in Chart 1. After 48-hour shaking culture at 27°C, the culture filtrate was adjusted to pH 3.0 with 6 N HCI and the precipitate was removed by centrifugation. The supernatant (24 liters) was adjusted to pH 5.0 with 6 N NaOH and passed through a column of carbon (6.5 x 20 cm). After the column was washed with 5 liters of distilled water, the adsorbed forphenicine was eluted with 50 % ethanol. The active eluate (3.32 liters) was concentrated to 25 ml under reduced pressure and subjected to DEAE-Sephadex A-25 (formate

Journal ArticleDOI
TL;DR: Alkaline phosphatase components in extracts of seven malignant and eleven benign ovarian tumors were characterized using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylalanine inhibition and reactivity against antiplacental ALP antiserum.

Journal ArticleDOI
TL;DR: The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles andAdenylATE cyclase and hormone-binding levels.
Abstract: The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity and 5′-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATPase on the one hand, and basal, hCG-stimulated, and fluoride-stimul...

Journal ArticleDOI
TL;DR: Previously demonstrated effects of NCS on DNA, such as the introduction of nicks not sealable by polynucleotide ligase, the release of thymine, and the formation of a malonaldehyde type compound, suggests that NCS-induced strand breakage involves base release accompanied by opening of the sugar ring with destruction of one or more nucleosides and results in a gap bounded by 3'- and 5'- phosphoryl termini.
Abstract: Neocarzionstatin (NCS)-induced strand breakage of DNA generates nonfunctional binding sites for the E. coli DNA polymerase I. Treatment of the NCS-nicked DNA with alkaline phosphatase at 65 degrees C prior to the polymerase reaction results in 60-100-fold stimulation of dTMP incorporation whereas in a control not treated with the drug there is only a 2-fold increase. Sites of strand scission on the NCS-treated DNA bear phosphate at the 3' termini. This conclusion is supported by the kinetics of release of inorganic phosphate from NCS-cut DNA by exonuclease III. Since our earlier work has shown that virtually all the 5' ends of the nicks caused by NCS bear phosphomonoester groupings, the 3'- and 5'- phosphoryl termini could be quantitated using alkaline phosphatase and exonuclease III. Over a wide range of drug levels the amount of inorganic phosphate released by alkaline phosphatase is approximately twice as much as that removed by exonuclease III, indicating the presence of equal amounts of 3'- and 5'- phosphoryl termini. This, taken together with other previously demonstrated effects of NCS on DNA, such as the introduction of nicks not sealable by polynucleotide ligase, the release of thymine, and the formation of a malonaldehyde type compound, suggests that NCS-induced strand breakage involves base release accompanied by opening of the sugar ring with destruction of one or more nucleosides and results in a gap bounded by 3'- and 5'- phosphoryl termini.

Journal Article
TL;DR: The localization of non-specific alkaline phosphatase activity during cleavage and blastocyst formation has been investigated in the mouse by electron microscopy and the significance of the differential localization of the enzyme is discussed, especially in relation to the differentiation mechanisms of the trophoblast and inner cell mass.
Abstract: The localization of non-specific alkaline phosphatase activity during cleavage and blastocyst formation has been investigated in the mouse by electron microscopy. The activityis detectable for the first time at the two-cell stage and is localized on the surface ofthe interblastomeric plasma membranes and on small cytoplasmic inclusions. It increases in the following stages, predominantly on the interblastomeric membranes, the outside membranes remaining devoid of reaction. From the four-cell stage on, small reactive grains are also observed in the crystalloid plates of the cytoplasm. At the morula stage, the plasma membranes of the inner mass cells are entirely marked by the reaction whereas the trophoblastic cells are polarized, with their inner surfaces positive and outside surfaces negative. At the blastocyst stage the enzyme is gradually eliminated from the membranes bordering the blastocoel and from the interblastomeric furrows of the trophoblast and primary endoderm. The significance of the differential localization of the enzyme is discussed, especially in relation to the differentiation mechanisms of the trophoblast and inner cell mass.

Journal Article
TL;DR: An increase in the level of serum alkaline phosphatase (ALP) may be caused by a wide variety of pathologic lesions that involve multiple organs, since the enzyme is an ubiquitous one.
Abstract: An increase in the level of serum alkaline phosphatase (ALP) may be caused by a wide variety of pathologic lesions that involve multiple organs, since the enzyme is an ubiquitous one. Before one attempts to identify a significant pathologic abnormality, the clinician should consider the possibility of a physiologic or spurious cause for an increased level of ALP. A decreased ALP level also has diagnostic value.

Journal ArticleDOI
TL;DR: In this article, a non-enzymatic method was used to extract matrix vesicle-enriched fractions from chicken epiphyseal cartilage, which achieved an additional 4-6 fold enrichment in alkaline phosphatase activity 16-30 fold purification.