Showing papers on "Alkaline phosphatase published in 1974"
TL;DR: In addition to the bone source, isoenzymes of alkaline phosphatase from liver, intestine and placenta have now been found to contribute importantly to the serum, either individually, or in combination, which provides new insights into the mechanism of uncompetitive inhibition.
400 citations
TL;DR: Rat liver microsomal fractions have been equilibrated in various types of linear density gradients and four groups of constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d.
Abstract: Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b(5), and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, beta-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.
366 citations
TL;DR: Liver homogenates have been submitted to quantitative fractionation by differential centrifugation and a final supernate (S) have been obtained, and the biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents.
Abstract: Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-beta-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.
309 citations
01 Jan 1974
TL;DR: In this article, the acid phosphatases in normal men and women have the highest activity with a broad optimum between pH 5.5 and 6.1, while prostatic phosphatase has a relatively high activity at pH 4.8.
Abstract: Publisher Summary This chapter elaborates on phosphatases that catalyze the hydrolytic cleavage of phosphoric acid esters. Alkaline phosphatases occur in practically all animal and human tissues. Acid phosphatases are also found in nearly all human and animal cells. The older methods of assay use the amount of phosphate liberated from a substrate as an index of phosphatase activity. The amount of 4-nitrophenol liberated per unit time, as determined in alkaline solution at 400–420 nm, is a measure of the phosphatase activity. The reaction is stopped by the addition of NaOH. The acid phosphatases in normal men and women have the highest activity with a broad optimum between pH 5.5 and 6.1. On the other hand the prostatic phosphatase has a relatively high activity at pH 4.8. The water content of nitrophenylphosphate is not constant in the commercial preparations and therefore should be checked. The buffer/substrate solutions should be stored at 0–4°C; they are stable for 1 week. The assay of phosphatase activity by continuous measurements has the advantage that only one pipetting is necessary and the measurements can be carried out rapidly.
278 citations
TL;DR: In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism, and incorporation of 3H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced.
274 citations
TL;DR: Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine, providing evidence not only for an asymmetrical distribution of the enzymes, but also for differences in the transport properties with respect to glucose.
174 citations
TL;DR: 4-Aminocatechol, an abnormal metabolite of phenacetin suspected of causing kidney damage, gave normal results for enzyme excretion suggesting the absence of renal lesions in the rat.
159 citations
TL;DR: It appears that gliadin is not directly toxic to GSE jejunal mucosa per se, but rather toxicity requires the participation of an endogenous effector mechanism which must first be stimulated in vivo.
Abstract: Jejunal biopsy specimens from patients with gluten-sensitive enteropathy (GSE) (obtained during gluten challenge) as well as from normal individuals and patients with other gastrointestinal abnormalities were cultured in vitro for 48 h in the presence or absence of a peptic-tryptic digest (P-T digest) of gliadin. In the absence of gliadin the alkaline phosphatase activity in the biopsy specimens obtained from normal control individuals increased from an initial value of 384 +/- 83 U to a 48 h value of 561 +/- 151 U (mean +/- SD) (difference significant at P < 0.01). The initial alkaline phosphatase activity of specimens obtained from patients with GSE was strikingly lower than that of normals, 117 +/- 79 U, and increased to a 48 h value of 399 +/- 203 U (difference significant at P < 0.01). The biochemical change in cultured biopsy specimens of GSE patients correlated with increases in the length and regularity of brush borders of epithelial cells as seen with the electron microscope. In the presence of a P-T digest of gliadin, the alkaline phosphatase activity of biopsy specimens of control individuals increased from an initial value of 384 +/- 83 U to a 48 h value of 578 +/- 156 U. In contrast, the alkaline phosphatase activity of biopsy specimens of patients with GSE in exacerbation showed a markedly diminished increase in activity during 48 h of culture; in this case the initial activity was 117 +/- 79 U and the final activity was 203 +/- 93 U. This inhibitory effect on increase of alkaline phosphatase activity during organ culture was specific in that a P-T digest of casein (a protein not toxic in vivo to patients with GSE) had no effect on alkaline phosphatase increases in culture. Finally, these results obtained with biopsy specimens taken from patients with GSE in exacerbation were compared with results obtained from patients with GSE in remission. Alkaline phosphatase activity of specimens obtained from the latter group of patients also increased during culture but in this instance P-T digest of gliadin in the culture medium had no significant inhibitory effect. In conclusion, the inhibitory effect of gliadin on intestinal epithelial cells in organ culture represents an in vitro model of gluten-sensitive enteropathy. Inasmuch as this effect of gliadin is not seen in cultures of specimens taken from patients in remission, it appears that gliadin is not directly toxic to GSE jejunal mucosa per se, but rather toxicity requires the participation of an endogenous effector mechanism which must first be stimulated in vivo.
156 citations
TL;DR: The results are consistent with the view that pyridoxal is an important transport form of the vitamin B 6 compounds, and indicate that a small amount of free PLP normally exists in equilibrium with the albumin-PLP complex.
148 citations
Journal Article•
TL;DR: Human blood neutrophilic leukocytes were separated and purified by modifications of the Hypaque/Ficoll and dextran separation methods, resulting in a suspension which was greater than 96% neutrophils, highly suggestive of a cytoplasmic membrane localization for this enzyme.
Abstract: Human blood neutrophilic leukocytes were separated and purified by modifications of the Hypaque/Ficoll and dextran separation methods, resulting in a suspension which was greater than 96% neutrophils. Neutrophils were prepared in 0.34 M sucrose containing heparin and were clarified of nongranular debris by sequential passage through polycarbonate filters of pore size 5 mu and 2 mu. Isopycnic sucrose gradients of such filtrates revealed three major bands. The gradient separated fractions were studied by electron microscopy including peroxidase cytochemistry and by enzyme assay for myeloperoxidase (MPO), beta-glucuronidase, muramidase alkaline phosphatase and acid phosphatase utilizing both p-nitrophenylphosphate (pnp) and beta-glycerophosphate as substrates. Peroxidase-positive granules were observed at both density 1.22 (band A) and density 1.20 (band B). Three peroxidase-negative granules were identified: the round or oval peroxidase-negative granule of density 1.22 (band A) and two smaller granules, distinguishable by size and shape at density 1.18 (band C). Band C granules contain crystalloid inclusions. Peaks of muramidase activity coincided with bands A and C, suggesting the presence of muramidase in the peroxidase-negative granules of density 1.22 and in one or both of the peroxidase-negative granules at density 1.18. beta-Glucuronidase was distributed like MPO, with a major peak in band B and a minor peak in band A. Acid beta-glycerophosphatase was largely in band A. Acid pnp phosphatase was nonspecifically associated with soluble nongranular protein which always remained at the origin of sucrose gradients. Alkaline phosphatase was not granule associated and sedimented alone to density 1.145, which is highly suggestive of a cytoplasmic membrane localization for this enzyme.
145 citations
TL;DR: After bacteriophage T7 infection, a protein kinase (EC 27137; ATP:protein phosphotransferase) activity can be demonstrated in E coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone as discussed by the authors.
Abstract: After bacteriophage T7 infection, a protein kinase (EC 27137; ATP:protein phosphotransferase) activity can be demonstrated in E coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone The bond between phosphate and protein shows the characteristics of serine phosphate: it is stable in 1 N HCl (100 degrees ) and cleaved by 1 N KOH (37 degrees ) and by alkaline phosphatase treatment Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms Enzyme activity in uninfected cells is negligible Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1) Ribosomal proteins are phosphorylated in vivo and are substrates in vitro Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP
TL;DR: Induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol, which suggests that the butyric acid cleaved from dibutiesryL cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.
TL;DR: Purified rat liver alkaline phosphatase appears to be a zinc-containing enzyme, loss of which results in irreversible inactivation, and loss of activity after electrodialysis can be rapidly reversed by the addition of magnesium.
TL;DR: The 19F nuclear magnetic resonance spectrum of m-fluorotyrosine alkaline phosphatase contains resolved resonances corresponding to different chemical environments for each m-flurotyrosine in the folded protein this article.
Abstract: Alkaline phosphatase (EC 3.1.3.1) containing m-flurotyrosine has been prepared from E. coli grown in the presence of m-flurotyrosine. The kinetic properties of the m-fluorotyrosine enzyme measured with p-nitrophenylphosphate at pH 8.0 and dinitrophenylphosphate at pH 5.5 are essentially the same as those of normal alkaline phosphatase. However, the ability of the m-fluorotyrosine protein to refold active enzyme after acid denaturation, while unchanged at pH 5.8, was markedly decreased at pH 7.6. This result implies that the tyrosines must be in their protonated form for the protein to refold, reassociate, and take on zinc. The 19F nuclear magnetic resonance spectrum of m-fluorotyrosine alkaline phosphatase contains resolved resonances corresponding to different chemical environments for each m-fluorotyrosine in the folded protein. This demonstrates that 19F nuclear magnetic resonance spectroscopy of enzymes specifically labeled with 19F, even with enzymes as large as alkaline phosphatase (molecular weight, 86,000), will provide a very valuable probe for conformational changes in proteins.
TL;DR: Analysis of the results established norms for several parameters and indicated areas of difference from results of other workers on the need for further biochemical studies of this nature.
Journal Article•
TL;DR: The results indicate that AAP and g-GTP are components of the outer membrane surface of the kidney proximal tubules, and are of potential value for diagnostic purposes.
TL;DR: Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH(4))(2)SO(4) to provide a way of controlling hydrophobic affinity chromatography.
Abstract: Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH4)2SO4. Retention on these columns was greatest at the highest concentrations of (NH4)2SO4. By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH4)2SO4-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine–Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.
TL;DR: Plasma membranes purified from cultured thyroid cells represent an excellent material to study the molecular events linked to receptor activation and the relation between thyrotropin concentration and adenylate cyclase activation of membranes was studied in time conditions of equilibrium binding of thyrotroropin.
Abstract: Plasma membranes have been prepared from porcine thyroid cells stimulated by dibutyryl adenosine 3′:5′-monophosphate in culture These cells show the histiotypic organization in follicles and the metabolic properties of gland follicular cells The plasma-membrane fractions have a density in sucrose of 118 g/ml (about 80%) and 116 g/ml (about 20%) The yield is 2 mg per 6 × 108 cells Electron microscopy showed vesicular structures and sheets of membranes often joined by membrane junctions Both showed the triple layer characteristic of the unit membrane ultrastructure Less than 3% of contamination by microsomes was derived from the assay of NADPH-cytochrome c reductase and NADH oxidase No contamination by mitochondria was indicated from succinate dehydrogenase activity measurements NaF-and thyrotropin-stimulated adenylate cyclase, 5′-nucleotidase and alkaline phosphatase are concentrated in the plasma-membrane preparations
Specific binding of 125I-labeled thyrotropin of high specific radioactivity (25 Ci/μmol) and preserved biological activity was compared to that in intact cells and to the plasma membranes from which they derived The rate constants of the thyrotropin association with cell or membrane (056 and 011 nM−1 min−1, respectively) and dissociation (036 and 025 min−1, respectively) were measured independently at 35°C (cells) and 27°C (membranes) The association constants derived from these rate constants (15 and 04 nM−1 for cells and membranes) are very similar to that (19 and 06 nM−1) determined independently from equilibrium data Therefore plasma membranes purified from cultured thyroid cells represent an excellent material to study the molecular events linked to receptor activation
The relation between thyrotropin concentration and adenylate cyclase activation of membranes was studied in time conditions of equilibrium binding of thyrotropin The semilogarithmic plot of thyrotropin concentration correlates sigmoidally with the percentage of adenylate cyclase activation Half-maximum stimulation is obtained for a thyrotropin concentration of 18 nM which correlates remarkably with the dissociation constant of 178 nM determined for the binding of the hormone to membranes
TL;DR: Findings suggest that specialized, morphologically different reticulum cells may be the “guide rails” for the different freely circulating lymphocyte populations.
Abstract: The capacity of certain B-lymphocytes to bind complement (demonstrated by erythrocyte-antibody-complement complexes, EAC) was used as a marker of the B-lymphocyte regions of the human white splenic pulp. This was carried out on cryostat sections in order to correlate enzyme histochemical findings (5-nucleotidase, ATPase, acid phosphatase, nonspecific esterase, alkaline phosphatase) to immunological functions. EAC were typically found in the follicle centers and marginal zone, whereas periarteriolar lymphocyte sheaths remained negative. The enzyme histochemical patterns of lymphocytes and reticulum cells allowed a clear distinction between areas with and those without EAC-binding. This was shown most clearly when the following enzymes were demonstrated in combination: 5-nucleotidase (5-Nase) + alkaline phosphatase, ATPase + acid phosphatase, and nonspecific esterase + acid phosphatase. 5-Nase correlated best to EAC-positive areas with a positive reaction in follicle wall lymphocytes and dendritic reticulum cells, whereas periarteriolar sheaths contained no 5-Nase-positive structures. Reticulum cells around periarteriolar lymphocyte sheaths at the margin of the red splenic pulp showed a particularly strong alkaline phosphatase reaction. The reticulum cells of the areas containing B-lymphocytes and those of the EAC-negative periarteriolar regions, which probably contain T-lymphocytes, were specifically labeled for different sets of enzymes. These findings suggest that specialized, morphologically different reticulum cells may be the “guide rails” for the different freely circulating lymphocyte populations.
Journal Article•
TL;DR: Investigation of the incidence of placenta-like alkaline phosphatase activity in serum of patients with tumors of the female breast and genitourinary tract suggests that the test may be useful in these conditions as an indicator of disease activity.
Abstract: SUMMARY The incidence of placenta-like alkaline phosphatase activ ity in serum was investigated in patients with diagnosed active malignant disease, in patients with a variety of nonmalignant conditions, and in normal individuals. The increased occurrence of this isoenzyme in cancer was not great enough to provide the basis for a useful diagnostic screening test. However, significant association of placenta like alkaline phosphatase activity in the serum of patients with tumors of the female breast and genitourinary tract was noted and suggests that the test may be useful in these conditions asan indicatorof disease activity.
TL;DR: The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes, while 5'-Nucleotidase was not localized to any marked extent in D. discoideum plasma membrane.
Abstract: A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N2 and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [125I]iodide. 5′-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH–cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH–cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.
TL;DR: It is demonstrated that the purified endonuclease causes single strand nicks to appear in ultra violet- and acetylaminofluorene-bound double stranded DNA.
TL;DR: A survey of placental alkaline phosphatase phenotypes in 5000 placentae of Caucasian, Negro and Asiatic Indian origin is described, with particular reference to the incidence, genetics and biochemical characteristics of the uncommon phenotypes.
Abstract: Placental alkaline phosphatase is a tissue-specific enzyme which has been shown to be polymorphic (Boyer, 1961 ; Robson & Harris, 1965). In all populations so far described there are three alleles with frequencies greater than 0.01, Play Plf and PI', though the particular gene frequencies differ considerably between populations (see Lucarelli-Palmarino et al. 1970, for review). In addition there are about 2 yo of unusual phenotypes which appear to be due to heterozygosity for a rare allele and one or other of the three common alleles. In this paper we shall describe a survey of placental alkaline phosphatase phenotypes in 5000 placentae of Caucasian, Negro and Asiatic Indian origin, with particular reference to the incidence, genetics and biochemical characteristics of the uncommon phenotypes.
TL;DR: A new model for the regulation of alkaline phosphatase biosynthesis is suggested, a ternary complex composed of phoB(+) and phoR(+) gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.
Abstract: Induction of alkaline phosphatase in wild-type Escherichia coli K-12 leads to the appearance of three new proteins in addition to alkaline phosphatase in the periplasmic space of the bacteria. These proteins are detected in autoradiograms of sodium dodecyl sulfate-acrylamide gel electropherograms of extracts from cells labeled with [35S]methionine. Studies with constitutive mutants defective in the three genes phoS, phoT, and phoR that have been shown to regulate alkaline phosphatase synthesis indicate that the three periplasmic proteins are coregulated with alkaline phosphatase. A mutant that has a deletion in the alkaline phosphatase structural gene phoA produces the three proteins, but a newly discovered mutant phoB that has a defect in the expression of alkaline phosphatase fails to produce the three proteins. phoB mutants are shown here to be unable to make detectable amounts of alkaline phosphatase polypeptides, as measured by immunoprecipitins or acrylamide gel electropherograms. On the basis of these results we suggest a new model for the regulation of alkaline phosphatase biosynthesis. In this model, a ternary complex composed of phoB+ and phoR+ gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.
TL;DR: There is an abnormal isoenzyme in the serum from patients with extrahepatic biliary obstruction and it is suggested that it may be associated with a lipoprotein carrier.
Abstract: The two main alkaline phosphatase isoenzymes in serum from patients with extrahepatic biliary obstruction have been purified. Properties of these isoenzymes, such as electrophoretic mobility, separation on gel filtration, ultracentrifugation characteristics, Michaelis constants, and sensitivity to neuraminidase have been studied and compared with the isoenzymes of liver and bile. The results show that there is an abnormal isoenzyme in the serum from these patients and that this isoenzyme is similar but not identical with the main bile isoenzyme. It is suggested that it may be associated with a lipoprotein carrier.