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Showing papers on "Alkaline phosphatase published in 1977"


Journal ArticleDOI
TL;DR: The results indicate that acid phosphatase is predominant in acid soils and that alkaline phosphatased are predominant in alkaline soils.
Abstract: Most studies on phosphatase activity in soils have been concerned with acid phosphatase. This study was conducted to determine the activity of phosphomonoesterases (acid and alkaline phosphatases), phosphodiesterase, and “phosphotriesterase”. The results indicate that acid phosphatase is predominant in acid soils and that alkaline phosphatase is predominant in alkaline soils. With universal buffer, the pH optima of phosphodiesterase and phosphotriesterase were at pH 10. The activities of these phosphatases in soils were much lower than those of the acid and alkaline phosphatases. Studies on the effects of various soil treatments on the activity of phosphatases in soils indicated that air-drying increased the activity of acid phosphatase and phosphotriesterase, decreased the activity of alkaline phosphatase, but did not affect the activity of phosphodiesterase. Steam sterilization of soils at 121 C for 1 h inactivated alkaline phosphatase, phosphodiesterase, and phosphotriesterase, but did not completely inactivate acid phosphatase. Addition of toluene to the incubation mixture did not markedly affect the activity of acid phosphatase, alkaline phosphatase, phosphodiesterase, but increased the activity of phosphotriesterase in soils. Studies of the kinetic parameters of phosphatases in the soils studied showed that the Km values ranged from 1.11 to 3.40 m m for acid phosphatase. from 0.44 to 4.94 m m for alkaline phosphatase, and from 0.25 to 1.25 m m for phosphodiesterase. Expressed as μg p-nitrophenol released·h−1·g−1 soil, the Vmax values ranged from 200 to 625 for acid phosphatase, from 124 to 588 for alkaline phosphatase, and from 46 to 127 for phosphodiesterase. The substrate of phosphotriesterase (tris-p-nitrophenyl phosphate) is insoluble in water; hence, the Km and Vmax values of this enzyme in soils could not be determined.

1,037 citations


Journal ArticleDOI
TL;DR: Evidence is presented that alkaline phosphatase treatment of human platelet beta-glucuronidase abolished its "high-uptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.
Abstract: Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is specifically taken up from the culture medium by human fibroblasts. Prior work has indicated that the enzyme exhibits charge heterogeneity and that "high-uptake" forms, i.e., those rapidly internalized by human fibroblasts, are more acidic than slowly internalized forms. Here we present two lines of evidence that the acidic group required for the high-uptake property of certain forms of the enzyme is a phosphate on, or in proximity to, a D-mannose-type carbohydrate. The first line of evidence was obtained from analysis of inhibition of enzyme pinocytosis by yeast mannans, phosphorylated sugars, and sugars. Mannans that contained phosphate were more potent inhibitors than those that did not contain phosphate. D-Mannose 6-phosphate was a more potent inhibitor than either D-mannose 1 phosphate or 2-deoxy-D-glucose 6-phosphate. D-Mannose and certain related sugars were weak pinocytosis inhibitors, while 2- and 4-epimers of mannose were noninhibitory. Competitive inhibition was demonstrated and the apparent Kis estimated for the following compounds: Saccharomyces cerevisiae mannan from mutant X2180-mnnl, 3 X 10(-6) M; mannan from wild-type S. cerebisiae, 3 X 10(-5) M; D-mannose 6-phosphate, 6 X 10(-5) M; L-fucose, 4 X 10(-2) M; and D-mannose, 6 X 10(-2) M. The second line of evidence comes from the observation that alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] treatment of human platelet beta-glucuronidase abolished its "high-uptake" activity, without diminishing its catalytic activity, and converted some forms of the heterogeneous enzyme to less acidic forms.

547 citations


Journal ArticleDOI
01 Nov 1977-Cell
TL;DR: Preliminary experiments suggest that even the "low uptake" form of α-l-iduronidase may be taken up by receptor binding, although with much lower affinity.

331 citations


Journal ArticleDOI
TL;DR: In this paper, the authors evaluated the effect of trace elements on acid and alkaline phosphatase activity in soil and found that all trace elements inhibit the activity of phosphatases in soils.
Abstract: Studies to evaluate the effects on the activity of acid and alkaline phosphatases of 20 trace elements showed that all trace elements inhibit the activity of phosphatases in soils. Results showed that the relative effectiveness of the trace elements in inhibition of phosphatase activity depends on the soil. When the trace elements were compared by using 25 ..mu..mole/g soil, the average inhibition of acid phosphatase in three soils showed that Hg(II), As(V), W(VI), and Mo(VI) were the most effective (average inhibition >50%) and that Ba(II), Co(II), and As(III) were the least effective (average inhibition <10%) inhibitors. Other elements that inhibited acid phosphatase activity in soils were: Cu(I), Ag(I), Cd(II), Cu(II), Zn(II), Mn(II), Sn(II), Ni(II), Pb(II), Fe(II), Cr(III), Fe(III), B(III), Al(III), V(IV), and Se(IV); their degrees of effectiveness varied in the three soils used. The most effective inhibitors of alkaline phosphatase activity in soils were Ag(I), Cd(II), V(IV), and As(V). All the trace elements that inhibited acid phosphatase activity also inhibited alkaline phosphatase activity. The order of effectiveness of the trace elements in inhibition of acid phosphatase activity was different from that of inhibition of alkaline phosphatase activity. Generally, the inhibitory effect of trace elements was less in the presence ofmore » 2.5 ..mu..mole/g soil than in the presence of 25 ..mu..mole/g soil. In addition to the effect of trace elements, phosphate ion inhibited acid and alkaline phosphatase activities in soils. Related anions such as NO/sub 2//sup -/, NO/sub 3//sup -/, Cl/sup -/, and SO/sup 2 -//sub 4/ were not inhibitory in the presence and absence of buffer.« less

215 citations


Journal ArticleDOI
TL;DR: Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.
Abstract: Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.

193 citations


Journal ArticleDOI
TL;DR: Data indicate that phosphate is a necessary component of the recognition marker on the enzyme for pinocytosis by human fibroblasts, and suggest that the phosphate on high-uptake forms of the enzyme is present as a phosphohexosyl moiety.
Abstract: We recently presented data showing that mannose-6-phosphate was a potent competitive inhibitor of pinocytosis of human platelet beta-glucuronidase, and that treatment of "high-uptake" forms of the enzyme with alkaline phosphatase destroyed the high-uptake property of the enzyme without diminishing its catalytic activity. These data indicate that phosphate is a necessary component of the recognition marker on the enzyme for pinocytosis by human fibroblasts, and suggest that the phosphate on high-uptake forms of the enzyme is present as a phosphohexosyl moiety. Results presented here show that mannose-6-phosphate is also a potent inhibitor of pinocytosis of the following enzyme preparations: (a) beta-glucuronidase from human spleen, liver, placenta, and urine; (b) beta-hexosaminidase and beta-galactosidase from human platelets; (c) beta-hexosaminidase from human fibroblast secretions. Alkaline phosphatase treatment of all these enzymes except beta-galactosidase, which was unstable to the incubation conditions and could not be tested, greatly diminished the uptake activity of the enzymes without diminishing their catalytic activity. These results suggest that phosphohexosyl recognition is a general characteristic of pinocytosis of lysosomal glycosidases.

166 citations


Journal ArticleDOI
TL;DR: The presumed precursor can dimerize to form active enzyme without being processed, and the resultant enzyme appears to be more hydrophobic than the mature enzyme.
Abstract: Alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] of E. coli was synthesized in a cell-free system, and the size of the direct translation product was analyzed. The product has a higher molecular weight than the mature alkaline phosphatase found in the periplasm. The direct translation product can be processed to the mature size by an E. coli membrane fraction; the processing activity copurifies with the outer-membrane fraction. The presumed precursor can dimerize to form active enzyme without being processed, and the resultant enzyme appears to be more hydrophobic than the mature enzyme. These findings are discussed in connection with the "signal hypothesis" proposed for the excretion of proteins across membranes.

159 citations


Journal ArticleDOI
TL;DR: Observations are consistent with the proposal that the glucocorticoid receptor can be inactivated by dephosphorylation and that only the phosphorylated form of the molecule is capable of binding steroid.
Abstract: Highly purified alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from calf intestine inactivates the glucocorticoid-binding capacity of soluble preparations from mouse fibroblasts (L cells) and rat liver. The unbound receptor is sensitive to inactivation whereas the steroid-bound receptor is unaffected. The ability of the enzyme preparation to inactivate the receptor, like its ability to dephosphorylate p-nitrophenyl phosphate, is dependent on zinc and inhibited by arsenate. Both the dephosphorylating and receptor inactivating activities coelute during DEAE-cellulose purification of the enzyme. There is no detectable proteolytic activity in the purified alkaline phosphatase preparation. In a mixed system containing both glucocorticoid and estrogen receptors, the glucocorticoid receptor is selectively inactivated. Although these observations do not prove that the receptor molecule itself is the substrate, they are consistent with the proposal that the glucocorticoid receptor can be inactivated by dephosphorylation and that only the phosphorylated form of the molecule is capable of binding steroid. A phosphorylation-dephosphorylation mechanism may be responsible for determining the level of active receptor in the cell.

152 citations


Journal ArticleDOI
TL;DR: A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs with gamma-32-P-ATP and T4 polynucleotide kinase.
Abstract: A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.

135 citations


Journal ArticleDOI
TL;DR: The plasmaαHS-glycoprotein concentrations of patients with Paget's disease of bone were shown to be substantially lower than the normal range, with significant negative correlation between the αHS- glycoprotein concentration and the plasma alkaline phosphatase activity.
Abstract: The spectrum of plasma proteins present in human cortical bone and permanent dentine has been determined. One plasma glycoprotein, theαHS-glycoprotein, was found to be present at a high concentration in both bone and dentine and was shown to be concentrated in the mineralized tissues with respect to the other plasma proteins by factors of between 30 and 100. In this respect theαHS-glycoprotein is analogous to the G2B-glycoprotein and α-glycoprotein of bovine and rabbit b one, respectively. The binding ofαHS-glycoprotein and albumin to calcium phosphates generated within serum samples has been studied at different serum: precipitate ratios. In each case all theαHS-glycoprotein was removed from the samples and theαHS-glycoprotein was concentrated with respect to albumin by factors ranging from 370 at the highest serum: precipitate ratio to 25 at the lowest ratio. The plasmaαHS-glycoprotein concentrations of patients with Paget's disease of bone were shown to be substantially lower than the normal range, with significant negative correlation between theαHS-glycoprotein concentration and the plasma alkaline phosphatase activity.

130 citations


Journal ArticleDOI
TL;DR: This work provides direct evidence that (i) secreted proteins thread through the membrane as growing peptide chains; and (ii) membrane-associated polysomes in bacteria are functionally attached to membrane and not merely trapped on disruption of the cell.
Abstract: To provide direct evidence for the hypothesis that secreted proteins may traverse membranes as growing chains, we labeled spheroplasts of Escherichia coli with a reagent (acetyl[35S]methionyl methylphosphate sulfone) that reacts with amino groups but does not cross the membrane. After fractionation, about 6% of the label in the membrane-polysome fraction was found to be attached to the polysomes. This attachment was via peptidyl-tRNA, as shown by several tests: release of most of the label from purified polysomes at low Mg2+; subsequent loss of about 25,000 daltons on cleavage by dilute alkali; release by puromycin; and release, accompanied by a marked increase in average molecular weight, on peptide chain completion. Moreover, a significant fraction of the completed chains was identified serologically and by molecular weight as a major periplasmic protein, alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1]. This work provides direct evidence that: (i) secreted proteins thread through the membrane as growing peptide chains; and (ii) membrane-associated polysomes in bacteria are functionally attached to membrane and not merely trapped on disruption of the cell.

Journal ArticleDOI
TL;DR: A sandwich non-competitive enzyme-immunoassay procedure using antigen or antibody cavalently linked to magnetic polyacrylamide-agarose beads has been developed and best results were obtained with alkaline phosphatase labelled antibodies.

Journal ArticleDOI
TL;DR: It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase, dependent on magnesium or calcium ions.
Abstract: A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 ×g supernatant is described. Preincubation of a crude enzyme extract with [γ-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 ± 280 only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [α-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 ×gsupernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and magnesium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ions was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.

Journal ArticleDOI
TL;DR: Observations give evidence that shedding of whole plasma membrane fragments (koinozymic shedding) is a widespread feature of viable cells.

Journal ArticleDOI
TL;DR: Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphate activity is synthesized.
Abstract: Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.

Journal ArticleDOI
TL;DR: In this paper, the binding of magnesium is dependent both upon pH and zinc content, and the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of Magnesium to alkaline phosphatase.
Abstract: Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.

Journal ArticleDOI
TL;DR: It appears that increased activities of maltase and alkaline phosphatase in the urine could be used as noninvasive, nondestructive indexes of renal damage, but may be no more sensitive than methods currently available.

Journal ArticleDOI
TL;DR: Deoxycholate, a dihydroxy bile salt, therefore appears to cause greater perturbation of membrane structure than the trihydroxybile Salt, cholate, and its conjugates.

Journal ArticleDOI
TL;DR: Allergen B previously isolated from honeybee venom and shown to be a mildly acidic protein consisting of polymers of a chain of 49,000 d is shown to have acid phosphatase activity and is homogeneous by several criteria.
Abstract: Allergen B previously isolated from honeybee venom and shown to be a mildly acidic protein consisting of polymers of a chain of 49,000 d is shown to have acid phosphatase activity. Allergen B is homogeneous by several criteria. No acid phosphatase, alkaline phosphatase, or esterase activity was found in any other allergen or fraction of bee venom. Acid phosphatase activity was also found in yellow jacket venom and extracts of venom sacs from bumblebees and paper wasps.

Journal ArticleDOI
TL;DR: The time of appearance and the ultrastructural localization of the enzyme activity of alkaline phosphatase, 5′-nucleotidase, Mg 2+ -ATPase, transport ATPase, cyclic AMP phosphodiesterase, cAMP-PDase, and adenylate cyclase were investigated in unfertilized eggs and in mouse preimplantation embryos.

Journal Article
TL;DR: The embryonal carcinoma cell line PCC4-F was shown to differentiate in vitro to a variety of cell types including cardiac and skeletal muscle, fibroblasts, ciliated and nonciliated epithelial cells, endodermal yolk sac cells, neurally derived cells, and fat cells.
Abstract: Summary The embryonal carcinoma cell line PCC4-F was shown to differentiate in vitro to a variety of cell types. A combination of light and electron microscopy and histochemical methods identifies or strongly suggests the presence of cardiac and skeletal muscle, fibroblasts, ciliated and nonciliated epithelial cells, endodermal yolk sac cells, neurally derived cells, and fat cells. Two cell lines were isolated from differentiated cultures. Preliminary comparisons have been made between these cell lines and the undifferentiated embryonal carcinoma cells. The derived cell lines produce an extracellular fibrous substance that gives a positive reaction to the periodate-Schiff reagent indicating a glycoprotein nature. There are significant differences in the incorporation of d-[3H]glucosamine and l-[3H]fucose into soluble extracellular proteins when the derived cell lines are compared with the embryonal carcinoma. These extracellular glycoproteins fall within narrow size classes. Enzyme studies indicate the presence of a new lactate dehydrogenase isozyme which is absent or greatly diminished in the derived cell lines. The specific activity of alkaline phosphatase is dramatically decreased in the derived cell lines, whereas those of acid phosphatase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase were not significantly different. Comparison of the derived lines with the properties of parietal endodermal yolk sac cells described by Lehman et al. (J. Cellular Physiol., 84: 13–28, 1974) suggests that these new lines are very similar if not identical in properties.

Journal ArticleDOI
TL;DR: The homogeneous enzyme is shown to be a sialoglycoprotein in nature and shows pyridoxal phosphate phosphatase activity along with p‐nitrophenylphosphate phosphate activity, which behave as mutual alternate competitive substrates.
Abstract: — Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion-exchange chromatography on DEAE-cellulose, and on DEAE-Sephadex followed by Sephadex G-200 filtration. By these steps, the enzyme is purified 22,920-fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p-nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described.

Journal ArticleDOI
TL;DR: It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PPi, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.
Abstract: Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg2+, Ca2+, and PPi. Further, the ATPase activity was not activated by Ca2+ in the presence of an optimal Mg2+ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PPi, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.

Journal ArticleDOI
TL;DR: The data arc consistent with a detoxification role for the glutathione S-transferases in intestine is consistent with that of liver.

Journal ArticleDOI
TL;DR: The evidence indicates a wider enzymatic and functional diversity among established human haemopoietic cell lines than has hitherto been suspected.

Journal ArticleDOI
TL;DR: The properties of three phosphatases from Salmonella typhimurium have been examined and it is found that the nonspecific acid phosphatase is a dimer of 27,000-dalton subunits, and the enzyme possesses activity towards hexose phosphates as well as other sugar phosphates.
Abstract: The regulation of three Salmonella typhimurium phosphatases in reponse to different nutritional limitations has been studied. Two enzymes, an acid hexose phosphatase (EC 3.1.3.2) and a cyclic phosphodiesterase (EC 3.1.4.d), appear to be regulated by the cyclic adenosine 3' ,5'-monophosphate (AMP) catabolite repression system. Levels of these enzymes increased in cells grown on poor carbon sources but not in cells grown on poor nitrogen or phosphorus sources. Mutants lacking adenyl cyclase did not produce elevated levels of these enzymes in response to carbon limitation unless cyclic AMP was supplied. Mutants lacking the cyclic AMP receptor protein did not produce elevated levels of these enzymes in response to carbon limitation regardless of the presence of cyclic AMP. Since no specific induction of either enzyme could be demonstrated, these enzymes appear to be controlled solely by the cyclic AMP system. Nonspecific acid phsphatase activity (EC 3.1.3.2) increased in response to carbon, nitrogen, phosphorus, or sulfur limitation. The extent of the increase depended on growth rate, with slower growth rates favoring greater increases, and on the type of limitation. Limitation for either carbon or phosphorus resulted in maximum increases, whereas severe limitation of Mg2+ caused only a slight increase. The increase in nonspecific acid phosphatase during carbon limitation was apparently not mediated by the catabolite repression system since mutants lacking adenyl cyclase or the cyclic AMP receptor protein still produced elevated levels of this enzyme during carbon starvation. Nor did the increase during phosphorus limitation appear to be mediated by the alkaline phosphatase regulatory system. A strain of Salmonella bearing a chromosomal mutation, which caused constitutive production of alkaline phosphatase (introduced by an episome from Escherichia coli), did not have constitutive levels of nonspecific acid phosphatase.

Journal ArticleDOI
TL;DR: Clustering of lymphocytes around Reed-Sternberg cells was noticed in single cell suspensions made from viable Hodgkin's lymphoid tissue, and Membrane receptor tests showed the lymphocytes surrounding the Reed- Sternberg cell to be T-cells.
Abstract: Clustering of lymphocytes around Reed-Sternberg cells was noticed in single cell suspensions made from viable Hodgkin's lymphoid tissue Cytocentrifugation of the suspension showed that clustering also occurred around a smaller cell type, thought to be the precursor of the classical Reed-Sternberg cell Time-lapse cine films taken of the clustering showed unceasing activity on the part of the lymphocytes migrating over the surface of the central cell Reed-Sternberg cells were reacted with anti-monocyte serum using indirect fluorescence techniques In its mature form at least, the Reed-Sternberg cell showed no activity with the antiserum No immunoglobulin was detected in the Reed-Sternberg cell using fluorescence techniques, but a few Reed-Sternberg cells showed diffuse cytoplasmic staining using the peroxidase-labelled antibody technique Membrane receptor tests showed the lymphocytes surrounding the Reed-Sternberg cell to be T-cells After proteolytic enzyme treatment to free lymphocytes from the surface, the Reed-Sternberg cell bound IgG-coated red blood cells indicating a probable Fc receptor Cytochemistry demonstrated weak non-specific esterase activity in a small minority of Reed-Sternberg cells, and absence of acid phosphatase, alkaline phosphatase and peroxidase A subpopulation of lymphocytes with distinctive segmentation of the nucleus was noted These were often to be seen participating in lymphocyte rosettes around the Reed-Sternberg cell

Journal ArticleDOI
TL;DR: A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures.
Abstract: A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.

Journal ArticleDOI
TL;DR: Optimum and specific assay systems have been developed which give linear kinetics for all enzymes and the range of enzyme activities in samples of human liver, obtained by closed needle biopsy, and sera have been determined.
Abstract: 1. Highly sensitive technique are described for the assay of plasma membrane (5'-nucleotidase, alkaline phosphatase), microsomal (neutral alpha-glucosidase, leucyl-2-naphthylamidase) and biliary canalicular (gamma-glutamyltransferase) enzymes and for nine acid hydrolases (acid phosphatase, phosphodiesterase, beta-glucosidase, alpha-glucosidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase, N-acetyl-beta-glucosaminidase, beta-glucuronidase) in human liver. 2. Optimum and specific assay systems have been developed which give linear kinetics for all enzymes. 3. The range of enzyme activities in samples of human liver, obtained by closed needle biopsy, and sera have been determined.

Journal ArticleDOI
11 Aug 1977-Nature
TL;DR: Ectopic synthesis of placental proteins, including the placental isoenzyme of alkaline phosphatase as well as the hormones hCG and human placental lactogen, may provide specific markers for neoplasm, since these proteins are not normally found in measurable amounts in the serum of men or of non-pregnant women.
Abstract: SOME human tumours and tumour-derived cell lines are capable of producing ‘eutopic’ peptide hormones and other proteins that are normally synthesised by the tissue from which the tumour originated. For example, choriocarcinoma cells, which are derived from cancer of the placental trophoblast, can synthesise human chorionic gonadotrophin (hCG) (refs 1, 2), and cultured pituitary adenomas can produce growth hormone (GH) (ref. 3). On the other hand, it is well established that a variety of malignant human tumours can produce ‘ectopic’ peptide hormones and other proteins which are not normally associated with the tissue of origin4–8. Ectopic synthesis of placental proteins, including the placental isoenzyme of alkaline phosphatase as well as the hormones hCG and human placental lactogen, may provide specific markers for neoplasm, since these proteins are not normally found in measurable amounts in the serum of men or of non-pregnant women9. Several human glycoprotein trophic hormones, including hCG, thyroid-stimulating hormone (TSH), luteinising hormone (LH), and follicle-stimulating hormone (FSH), are composed of two non-identical subunits, α and β (refs 10–13). These four hormones have virtually identical α subunits; in contrast each has a unique β subunit, which is responsible for biological specificity12. Complete hCG and free subunits are secreted by normal and neoplastic placenta1,2,14 and by many non-trophoblastic tumours15,16. Since hCG is a eutopic hormone for trophoblastic tumours, but an ectopic hormone for non-trophoblastic tumours, it occured to us that its synthesis by the two types of tumours might be regulated differently. We therefore compared the synthesis of hCG and hCG-α by three choriocarcinoma cell lines—BeWo (ref. 1), JEG-3 (ref. 2) and Reid (P. O. Kohler, unpublished observations)—with the synthesis by two non-trophoblastic tumour-derived cell lines—ChaGo and HeLa. (ChaGo was derived from an hCG-producing pulmonary carcinoma16, and HeLa from a carcinoma of the cervix17.) We also tested the effects of sodium butyrate on the synthesis, since this compound had been shown to induce hCG-α and hCG synthesis in certain HeLa cells18. We found that in the absence of inducer, all of the cell lines can produce hCG-α—the trophoblastic tumour cells at roughly comparable rate, but the non-trophoblastic tumour cells at widely different rates (Fig. 1).