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The substrate specificity and inhibition of alkaline phosphatases of cow's milk and calf intestinal mucosa
... With 250 mM PP i , the reaction is also faster at pH 8 but the reaction is very slow and reaches a steady level with no further hydrolysis (data not reported). This suggests a possible inhibition by the substrate PP i , as several authors already reported (Butterworth, 1968;Fernley & Walker, 1967;Morton, 1955;Nayudu & Miles, 1969). This is in contrast to the acid phosphatases PhoN-Sf and PhoN-Se, which completely consume PP i under similar conditions and which are not inhibited by PP i (Tanaka et al., 2003;van Herk et al., 2005). ...
... The enzyme was completely inactive in 100 mM P i . This suggests that 100 mM PP i will never be hydrolyzed completely, because P i will inhibit the alkaline phosphatase already at low concentrations as has already been reported by many authors (Morton, 1955;Portmann, 1957). ...
... Furthermore the AP is commercially available in contrast to the bacterial acid phosphatases. As shown by us PP i did not inhibit the alkaline phosphatase from bovine intestine, but the product of the reaction, P i , had a strong inhibitory effect on the activity of AP as found by many authors (Butterworth, 1968;Fernley & Walker, 1967; Morton, 1955;Nayudu & Miles, 1969). Already at 10 mM of P i the hydrolysis of PP i was inhibited for 10 % and at 25 mM P i 40 % inhibition was found. ...
Phosphatases (AP, E.C. 3.1.3.1) are hydrolytic enzymes that naturally hydrolyse phosphomonoesters but in a so-called transphosphorylation reaction these enzymes are also able to transfer a phosphate group from phosphorylated compounds to alcoholic functions. This transphosphorylation catalysed by acid phosphatases using pyrophosphate as a phosphate donor has been studied in some detail. However, the acidic pH optimum of these enzymes limits some of their applications. The catalytic features of alkaline phosphatase are similar to the acid phosphatases and its alkaline pH optimum suggests a possible application of this enzyme in phosphorylation reactions which need to be carried out at higher pH. Here we explore the synthetic potential of bovine intestine alkaline phosphatase (AP) in the phosphorylation of dihydroxyacetone (DHA) and glycerol using pyrophosphate (PPi) as phosphate donor. The phosphorylated compounds are intermediates in two multi-enzymatic cascade reactions for the synthesis of carbohydrates. The yields of dihydroxyacetone phosphate (DHAP) and glycerol-1-phosphate at pH 8 (2.6 mM and 2.2 mM, respectively) were comparable to the results obtained with the acid phosphatases at pH 4. Nevertheless, when the cascade reactions were carried out at pH 8, very low conversions were measured due to inactivation of the alkaline phosphatase by the product phosphate. To circumvent this inhibition, the alkaline phosphatase was immobilized on aldehyde-activated beads (Sepabeads EC-HA). The immobilization greatly diminished the inhibition by phosphate, and the immobilized alkaline phosphatase at pH 8 gave the same conversions in the cascade reaction starting from DHA as obtained with the acid phosphatase at pH 6. However, the immobilized enzyme was active for only one catalytic cycle and the beads could not be reused.
... A phosphatase well active with ΨMP and able to discriminate against Rib1P and Rib5P is not known. Commercial enzymes (e.g., calf intestine phosphatase) are not usable due to lack of specificity 83 . In a genome-wide study of E. coli phosphatases, Kuznetsova et al. analyzed the substrate spectrum of 23 enzymes of the structural superfamily of haloalkane dehalogenases. ...
As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine (U) by the corresponding C-nucleoside, pseudouridine (Ψ), in 1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me-Ψ−5’-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1N-5C rearrangement of β-d-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via d-ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ-5’-phosphate (ΨMP), gives Ψ. Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the ΨMP, but not from the intermediary ribose phosphates. Discovery of Yjjg as ΨMP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N-C rearrangement, the optimized U reaction yields a supersaturated product solution (∼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of ∼105 mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C-nucleoside synthesis in high efficiency.
... Purified phosphatase from the duodenal mucosa of the calf hydrolyses phenylphosphate about 2-4 times as fast as beta-glycerophosphate (Morton, 1955). Kato (1960) has observed that with the chick embryo duodenum the ratio of P liberated from phenylphosphate to P liberated from beta-glycerophosphate (PhP/bGlP) is not constant, but rises from 18 at 14 days to 4-3 at 17 days, and then falls to 2 4 at hatching. ...
The epithelium of the duodenal mucosa of vertebrates is strikingly uniform. In all but the cyclostomes and a few fishes, the epithelium is made up of tall columnar absorbing cells, with mucus-producing goblet cells interspersed. The columnar cells have a high cytoplasmic/nuclear ratio, with the cytoplasm arranged into an apical zone containing a prominent Golgi apparatus, and a basal zone. The apical surface is featured by a sheet of microvilli that are extremely rich in alkaline phosphatase. The attainment of these characteristics has been shown to take place in a brief critical period, just before hatching in the chick embryo (Moog, 1950) and just before birth in the mouse (Moog, 1951), although in the latter animal a second period of phosphatase accumulation precedes weaning. The guinea-pig has now been added to this comparative series, because the protracted gestation period (about 67 days) might permit some further insight into the problems of duodenal differentiation.
... The concentration of apo-Damino acid oxidase was 0.41 Units mL"1. (Martland and Robinson, 1927;Morton, 1955b). This inhibition is strictly competitive ( Lazdunski and Ouellet, 1962 ...
A simple to use, quantitative and extremely sensitive colorimetric assay, designed to be used in a detection system in diagnostic assay systems, is described. The technology of an amplified colorimetric assay, developed by London Biotechnology Limited, is based on a conceptually new principle which is covered by an issued generic patent. The enzyme amplification cascade detects alkaline phosphatase, a widely used enzyme label in immunoassays and gene probes, via dephosphorylation of a novel substrate, FADP, to produce the prosthetic group FAD. This binds stoichiometrically to inactive apo-D-amino acid oxidase, to produce the active holoenzyme which oxidizes D-proline to yield hydrogen peroxide; this in turn is quantitated by a coupled reaction utilizing horseradish peroxidase. The signal-to-noise ratios and sensitivity are enhanced by the kinetics of the amplification cascade. The criteria for the quality control of the reagents used the the cascade system are much more severe than those used in conventional assay systems. Methodologies for the monitoring of contaminants in the production of these materials have been developed. Thus the detection system itself has been used to trouble-shoot the production processes. The primary substrate, FADP, and apo-D-amino acid oxidase have been prepared in high yield and purified to the level required. FADP has been produced with a contamination level of FAD equivalent to 2 ppm and apo-D-amino acid oxidase has been prepared which contains only 10 ppb alkaline phosphatase activity. Evaluation of the technology has demonstrated it to be suitable for use on high quality clinical analyzers; and the methodology has been used in conjunction with the releasable linker technology developed by Du Pont. This system has been used to measure TSH across the entire range of clinical interest.
... Alkaline phosphatase has a broad specificity for phosphate esters of alcohols, amines, pyrophosphate, and phenols, and is routinely used to dephosphorylate proteins, peptides and nucleic acids. [38][39][40][41] Following treatment with the phosphatase, the phosphorylated tryptic linker peptide X 9 GGGGSGGGGpSR was no longer detectable by LC-MS (Fig. 6A), verifying the enzymatic removal of the phosphate group (XIC: z D 2 and 3, m/z windows 561.61-561.63 C 588.26-588.28 ...
Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G4S)2] of a fused heavy chain containing in total 2 (G4S)2-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins. © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC
... Moreover, the kinetics of a few phosphotransferases have a sigmoid relationship with concentrations of anionic cofactors at a fixed amount of Mg 2+ , but show Michaelis-Menten kinetics when the ratio Mg 2+ to anionic cofactor is maintained constant [149,168,169]. While on the one hand various types of complexes can be formed with a dynamic fraction as a function of the ratio of Mg 2+ :ATP 4− [161], only one configuration of the complexes such as Mg- ATP 2− for ATP:glycerol phosphotransferase [73] or MgPP i 2− for phosphatases [149,170] does act as the most bioactive form. There appears an optimum ratio of Mg 2+ to anionic cofactor for which such enzymes show maximum activity [73,149,153,168,169]. ...
This chapter focusses on selective biocatalytic phosphorylation reactions of metabolites. Phosphoryl group transfer reactions are of fundamental importance for living organisms and a variety of enzyme classes are involved in catalyzing these phosphorylation reactions. A large number of phosphorylases, phosphatases, and phosphotransferases/ kinases have been described as enzymes for the biocatalytic formation of new O-P- bonds, while the number of enzymes catalyzing the formation of N-P-, S-P-, and C-P-bonds is much smaller. Analytical method development, in particular separation and detection methodologies, for the direct analysis of biocatalytic phosphorylation reactions, are described as decisive steps for the assay and screening of novel phosphorylating biocatalysts, for investigating their functional properties and for process design. It is equally important for obtaining product stability data, as the areas of stability for the phosphorylated metabolites are best determined by experiments. As high-energy phosphate compounds are key players in life processes, the most important phosphate compounds are discussed in this chapter. Even though ATP is preferable in most enzymatic phosphorylation reactions, additional phosphoryl donors with varying energy level, availability, and specificity are of great interest. The stereo- and regioselectivity of phosphorylating enzymes offers benefits for the production of phosphate esters. A selection of new emerging phosphorylation reactions is shown. The subchapter on reaction engineering for biocatalytic phosphorylation processes provides basic reaction engineering principles, reaction kinetics, reaction system and process optimization strategies for biocatalytic phosphorylation reactions. The transition from stoichiometric phosphorylation reactions to biocatalytic phosphorylation reactions improves selectivity, safety, health, and environment, reduces the number of reaction steps in existing phosphoryla-tion routes, and decreases the waste-to-product ratio, if the phosphate donor is either recycled or used up in the reaction.
... Prelabeling of cells with [nP]orthophosphate confirmed the loss of phosphate during enzymatic treatment. Pyrophosphate, an inhibitor of alkaline phosphatase (27), blocked these effects of enzymatic treatment. This indicates that the enzyme-induced shift was due to changes in phosphorylation rather than to proteolysis by a contaminant in the preparation. ...
This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.
... The incomplete dephosphorylation was probably caused by the presence of sites with phosphoamino acids that were unavailable and/or not recognized by alkaline phosphatase. It is noteworthy that in natural conditions, there are plenty of phosphatases differing in their substrate specificity [17]. ...
Androgenesis represents one of the most important and fast-developing biotechnological approaches for the production of haploid and di-haploid crop plants with great potential for plant breeding. Tobacco serves as a suitable model plant with available protocols for androgenesis. However, it is necessary to understand the molecular regulatory mechanisms underlying this process. Phosphorylation is the most dynamic posttranslational modification. In a time, the cell contains only few percent of phosphoproteins that can furthermore be accompanied by their native forms. Thus, the enrichment techniques have to be applied in order to concentrate the phosphorylated forms. The aim of our studies was to compare the phosphoproteins extracted by TCA/acetone out of mature pollen and 30-min-activated pollen. The crude protein extracts were enriched by the metaloxide/hydroxide affinity chromatography (MOAC) with aluminium hydroxide matrix. The efficiency of the enrichment was verified by 1D SDS-PAGE of the fractions coming out of the enrichment. The separated phosphoproteins were detected by phospho-specific ProQ Diamond stain (Invitrogen) whilst total proteins were stained by colloidal CBB G250. In this short paper, we present our aims in revealing the MOAC specificity for phosphoprotein enrichment. The enrichment of dephosphorylated mature pollen crude extract and the original mature pollen crude extract were compared. Since alkaline phosphatase did not accomplish the dephosphorylation step properly, we decided to apply cerium dioxide for sample dephosphorylation. Since cerium dioxide captured the phosphoproteins rather than dephosphorylated them, the proof of MOAC specificity was impossible to be achieved by these experiments.
... In this latter case, experimental measurement of K M is probably underestimated. [28,29,30] Production of a non productive or inactive complex is taken into account for a new fitting procedure of experimental adsorption isotherm. Table 1. ...
Real time monitoring of electrolyte resistance changes during hydrolysis of 4-nitrophenylphosphate (pNPP) by alkaline phosphatase (ALP) bound on paramagnetic-beads was performed into a small dielectric channel. The reaction kinetic fit with a non-competitive substrate-inhibition equation. Michaelis-Menten apparent constant, KM(app), was determined as 0.33±0.06mM and the maximum apparent rate, Vmax(app) as 98±5pMs(-1). The detection limits were 15fM for ALP and 0.75mM for pNPP. This miniaturized device constitutes a powerful tool for analysis of interaction between ligands.
... The enzyme that we used also works on a broad class of compounds and can hydrolyze organic and inorganic polyphosphates as well as simple phosphomonoesters (Morton 1955;Fernley and Walker 1967). We have not attempted to distinguish between these substrates. ...
Abstmct Complex phosphorus compounds were classified functionally according to the manner by which orthophosphate was released. Filtrable phosphorus compounds in two cutrophic lakes (East and West Twin lakes) and a humic bog (Crazy Eddie Bog) in northeastern Ohio wcrc fractionated by anion-exchange and gel-filtration chromatography. Fractions were analyzed for soluble reactive phosphorus and total dissolved phosphorus; absorbance at 400 nm was used as a measure of filtrable "yellow acids." The cutrophic lakes contained numerous low molecular weight compounds which were resistant to low-dose ultraviolet irradiation but readily released orthophosphate upon treatment with alkaline phosphatase. Filtrable phos- phorus compounds of the humic bog were predominantly high molecular weight, cochro- matographed with the yellow acids in each fractionation procedure, and resisted enzyme hydrolysis, but released orthophosphate upon irradiation with low doses of ultraviolet light.
Enzymatic hydrolysis of phosphoglycolate proceeds through O-P bond cleavage as determined by reaction in H2¹⁸O and analysis of the trimethylsilyl derivatives of the reaction products by mass spectrometry. No phosphate, hydroxyl, or carboxyl exchange occurred. End product inhibition was consistent with an ordered release of products, first the alcoholic product, glycolate, then phosphate. Analysis of the data indicated that the phosphate enzyme complex dissociated very rapidly, and this was confirmed by use of alternative phosphomonoester substrates. Maximum velocity with these alternate substrates was found to be proportional to the pK, of the corresponding alcoholic product, indicating the rate-limiting step in the reaction was protonation of the bridge oxygen. The use of substrate analogs further suggested that enzymatic specificity resides in exacting steric requirements for binding, and that large alkyl groups were excluded on this basis. Phosphoglycolate phosphatase catalyzed transphorylation to a wide range of acceptors and was inhibited at the active site by diisopropyl-fluorophosphate. The data suggest that the reaction sequence proceeds via a phosphoenzyme intermediate.
N-Ethylmaleimide slowly inactivated the enzyme, the rate being greatly increased by P-glycolate, but not by magnesium or phosphate ions. The data suggest a conformational change is necessary to induce the transition state complex and phosphoenzyme formation. This may account for the phosphate acceptor specificity and is consistent with the failure to observe an enzyme-mediated H2O-phosphate oxygen exchange.
The antigenic distinctiveness of individual organ-specific isoenzymes of human alkaline phosphatase was shown by immunochemical means. A two-step method involving two separate antigen-antibody reactions was utilized to identify and measure the isoenzymes. The first reaction consisted of reaction of the isoenzyme with its antiserum; the second reaction consisted of precipitating the enzyme-antibody complex with anti-γ-globulin. The second reaction was necessary because the enzyme was neither inhibited nor precipitated by specific antibody. By this method, individual isoenzymes could be identified from mixtures of isoenzymes.
The results clearly show that antibody to liver phosphatase and antibody to placental phosphatase were specific for their respective homologous antigens, and did not react with alkaline phosphatase from bone, neutrophils, kidney, or intestine. Thus, there are at least three antigenic types of alkaline phosphatases: one derived from liver, one from placenta, and one or more from other organs.
Alkaline phosphatase has been extensively used in diagnosis during the past three decades, and there has been much discussion concerning the origin of the serum enzyme in hepatobiliary disease. It was formerly considered to originate mainly in the osteoblasts of bone and to be excreted in the bile (Gutman, 1959). Many other tissues including the liver, intestinal mucosa, kidney and placenta have been shown to contain the enzyme (Bodansky, 1937, 1948; Armstrong and Banting, 1935; Cloetens, 1939; Sherlock and Walshe, 1947; Burke, 1950; Ross, Iber and Harvey, 1956; Ahmed and King, 1959; Grossberg, Harris and Schlamowitz, 1961; Hodson, Latner and Raine, 1962; Fishman, Green and Inglis, 1962; Moss and King, 1962; and others, reviewed by Posen, 1967), and there is now strong evidence that other tissues, especially the liver, contribute to the serum alkaline phosphatase activity. A number of chromatographic and electrophoretic techniques have been applied in attempts to identify the tissue of origin of the serum enzyme activity, and these led to the recognition of its heterogeneity.
Uranium is a predominant radionuclide arising out of operations of mining, milling and fuel fabrication for nuclear power plants. While chemical and biological methods using several micro-organisms have been employed for the remediation of uranium from such effluents, use of immobilised enzymes for the purpose has not been reported. The current investigation deals with a purified calf-intestinal alkaline phosphatase which is capable of driving uranium precipitation in the presence of a phosphate-containing substrate. The enzyme, both in free form and in an immobilised form on polyvinylidene fluoride membrane, was investigated for the precipitation of uranium using para-nitrophenyl phosphate as substrate. The results indicated that while the enzyme in free form could precipitate most of the uranium within 6 h, the immobilised enzyme could achieve the same in 24 h. There was a spurt in the precipitation of uranium by the immobilised enzyme during the initial period of 3 h followed by a linear increase for the remaining period. The immobilised enzyme was found to be stable and catalytically active for 3 years in precipitating uranium. The results obtained offer polyvinylidene fluoride immobilised alkaline phosphatase from calf intestine as a new tool for the remediation of uranium from effluents. Further, evidence has been also shown that no requirement for any cellular architecture/scaffolding exists for uranium mineralisation to occur.
Plant tissues vary considerably in their content of protein and it may, therefore, be anticipated that some tissues, in, reference to others, may lend themselves more readily to enzyme isolation and purification. The leaves of the tapioca plant contain 20–36% of crude protein (Rogers, 1959), whereas the phylloclades of cactus have only about 7%, both on a dry weight basis (Sanwal, 1960). The nature of the nonprotein material of the starting material also influences the course of enzyme purification. The presence of mucilaginous material in tissues such as cactus interferes with conventional techniques of enzyme enrichment. Whereas, in general, the investigator is concerned with the enzyme-make up of a given tissue and preliminary screening does not find much scope, it is sometimes possible to choose a special tissue where the given enzyme is present in optimum concentration. In the animal tissue, high alkaline phosphatase activity seems to be associated with intestinal mucosa, acid phosphatase with kidney and spleen and, even more so, with prostate and 5′-nucelotidase activity with testis. Germinating seeds constitute a good sources of acid phosphatases (Newmark and Wenger, 1960). Plant tissue and microorganisms are, in general, poor sources of alkaline phosphatase, but Garen and Levinthal (1960) showed that when Escherichia coli was grown in a medium containing limiting amounts of orthophosphate, as much as 6% of the total protein synthesized was alkaline phosphatase. The availability of such a concentrated source facilitated the isolation of the phosphatase and study of its properties.
In this chapter, we first describe some of the compounds of phosphorus that can serve as substrates in the alkaline phosphatase reaction. We discuss some of their chemical characteristics, and their nonenzymatic hydrolysis. We then go on to examine the sequence of the alkaline phosphatase reaction step by step.
Alkaline phosphatase is a nonspecific phosphomonoester hydrolase widely found in prokaryotes, invertebrates, and higher animals; plants, though rich in nonspecific acid phosphatase activity, are not replete in alkaline phosphatase. The enzyme has long been recognized in mammals because of its ready detection even in whole tissue preparations by virtue of its high activity at alkaline pH (example, pH 10). In mammals, alkaline phosphatase is an essential, tissue-specific, membrane-associated enzyme. It is required for the deposition of calcium phosphate as hydroxyapatite in bone. The proliferating zone of epiphyseal plates is the richest source of alkaline phosphatase in bone. The enzyme has been implicated in the accumulation of calcium and phosphate by matrix vesicles where it is attached to the membranes of those vesicles. In the disease hypophosphatasia, because of a mutation in the structural gene for bone alkaline phosphatase, humans are deficient in this enzyme and show poor ossification and bone development.
This chapter will discuss some practical aspects of alkaline phosphatase assays. While the remarks are based predominantly on experience gained with human blood serum, many of the points raised are also applicable to the assay of alkaline phosphatases from other sources.
Alkaline phosphatase has been extensively used in diagnosis during the past three decades, and there has been much discussion concerning the origin of the serum enzyme in hepatobiliary disease. It was formerly considered to originate mainly in the osteoblasts of bone and to be excreted in the bile (Gutman, 1959). Many other tissues including the liver, intestinal mucosa, kidney and placenta have been shown to contain the enzyme (Bodansky, 1937, 1948; Armstrong and Banting, 1935; Cloetens, 1939; Sherlock and Walshe, 1947; Burke, 1950; Ross, Iber and Harvey, 1956; Ahmed and King, 1959; Grossberg, Harris and Schlamowitz, 1961; Hodson, Latner and Raine, 1962; Fishman, Green and Inglis, 1962; Moss and King, 1962; and others, reviewed by Posen, 1967), and there is now strong evidence that other tissues, especially the liver, contribute to the serum alkaline phosphatase activity. A number of chromatographic and electrophoretic techniques have been applied in attempts to identify the tissue of origin of the serum enzyme activity, and these led to the recognition of its heterogeneity.
The estimation of serum phosphatase, especially alkaline phosphatase, is one of the most frequently performed tests in clinical biochemistry. Alkaline phosphatases derive from a number of tissue sources, so that the activity in a given specimen of serum at a given point in time reflects several production rates and several biological decay rates. Skeletal alkaline phosphatase activity in the circulation rises only when there is a considerable increase in osteoblastic activity and sometimes not even then. This chapter describes a variety of methods for the study of the localization of skeletal alkaline phosphatase in various parts of bone, its association with different cells, and its intracellular distribution. Osteoblasts and osteocytes are rich in alkaline phosphatase; however, they contain little acid phosphatase. The reverse is true of osteoclasts. Enzymes are proteins whose presence is detected by their ability to catalyze certain reactions in vitro. Therefore, they are not quantitated in absolute units such as weight per volume but in units of activity.
During the last quarter of the 19th century and the early years of this century, several writers stressed the interconvertibility of organic and inorganic phosphate in living animals. Dalton1 pointed out that although compounds rich in organic phosphate comprise a significant part of the diet, none is found in excreta. The presence of phosphate in urine was attributed to the “oxidation” of organic phosphates. LeClerc and Cook2 measured urinary phosphate in dogs and rabbits and found identical values regardless of whether dietary phosphate consisted of wheat bran, egg yolk, or inorganic phosphate salts. Mathison*3 incubated glycerophosphate for “weeks” at 39°C with a number of enzymes, such as trypsin, pepsin, and fresh pancreatic juice, and found no hydrolysis. However, when “Subject M, aged 25” ingested glycerophosphoric acid, a prompt increase was noted in urinary inorganic phosphate.3 Gregersen4 showed that rats fed a synthetic diet containing inorganic phosphate could use this material to build up organic compounds. Fingerling6 demonstrated the same phenomenon in ducks. Several other groups7–9 showed that ingestion or injection of organic phosphate esters led to an increase in the excretion of inorganic phosphate in the urine. While it was generally realized that these substances were extremely stable in vitro,3,8–10 no particular hydrolytic enzyme was identified to account for the in vivo effects. It appears that many of the early workers believed that a lipase was responsible for the hydrolysis of phosphate esters.11
The identity of enzymes cannot be determined exactly as long as one enzyme is mixed with others of the same group.1
Most phosphorylated metabolites are derivatives of orthophosphoric acid. Phosphorylated metabolites are stable at about pH 7 in aqueous solution at temperatures of living organisms and this high kinetic stability associated with a large potential reactivity-as in the phosphoric acid anhydrides and in enol-pyruvate phosphate-might be a special feature of phosphates, which led to the widespread dependence of metabolism on phosphorylated compounds. With relation to bond cleavage, several mechanisms might be considered for the phosphohydrolases. It is possible that water acts freely from solution, attacking the enzyme-bound phosphorylated substrate. The water might be bound and attack the phosphorylated compound from a specific site on the enzyme surface. The phosphoryl-transfer reactions of alkaline and acid phosphatases indicate that a phosphorylated enzyme is formed as an intermediate, and this has been demonstrated with alkaline phosphatases from bone, intestinal mucosa, and Escherichia coli.
Sugars play important roles in many biological processes, such as nutrition, metabolism, cell structure formation, and immunological protection. Phenylboronic acid (PBA) derivatives have been widely used as recognition molecules for sugars based on the formation of boronate ester bonds between boronic acid and 1,2-diol or 1,3-diol of sugars. In this work, ferroceneboronic acid (FBA, an electrochemically active ferrocene derivative) was used to investigate the interaction between boronic acid and phosphated as well as non-phosphated sugars. Based on the difference in the voltammetric responses of the complexes between FBA and examined sugars, FBA probe was used to monitor the activity of enzymes, such as phosphoglucose isomerase (PGI) and alkaline phosphatase (ALP). This work would be valuable for understanding of the diol-boronate interaction and screening of new enzyme inhibitors.
Isoenzyme refers to a biochemically distinct class of catalytically active proteins with the same specificity of bond cleavage or alteration, which can occupy several zones following electrophoresis. The chapter focuses on the mechanism of catalysis and uncompetitive inhibition and provides analyses of studies reporting distinctive immunochemical properties as well as physical properties of enzymes prepared from several organs. In the case of alkaline phosphatase, the studies rely on a combination of organ source, biochemical properties, and electrophoretic distinctions as guidelines for working in this field. Placental alkaline phosphatase is an isoenzyme of alkaline phosphatase, which can be distinguished from other alkaline phosphatases by biochemical means. The different molecular forms of this isoenzyme, which can be produced and separated by physical means, are referred as variants of placental alkaline phosphatase. Hyperphosphatasemia in diseases of liver and bone has made the serum alkaline phosphatase determination the most frequently demanded enzyme assay.
Adenosine 5′-monophosphate (AMP) can be attached to Merrifield resin [Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149–2154; commercially available as Bio-Beads SX-2, Bio-Rad Laboratories, Richmond, Calif., U.S.A.] via a covalent linkage which subsequently can be cleaved under non-destructive conditions. The polymer-bound AMP (Compound I) in pyridine reacts with anhydride (Compound II) to produce polymer-bound 5′-monophosphate(Compound III). Similarly, the [1-14C]glycine analog of Compound III and the related cytidine 5′-monophosphate (CMP) derivatives have been prepared. The relative ease of preparation of these compounds indicates that glycine is more reactive than phenylalanine and that both amino acids combine with adenosine more readily than with cytidine. The resemblance between these polymer adducts and the acceptor terminus of a tRNA is such that the observed differences in reactivity as measured even in pyridine may be important in terms of molecular selectivity and chemical evolution.
Bovine milk contains a large number of enzymes (about 60 have been identified) which originate from the mammary gland tissue cells, blood plasma and blood leucocytes. Although they represent a minor fraction of the total milk protein, they have received in-depth scientific investigation over the past 50 years. Several major reviews1–6 have been published during this period, together with hundreds of papers on detailed scientific studies on many of these individual enzymes.
Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein‐stabilizing effect during freeze–thawing and freeze‐drying. Six enzymes, different in terms of tolerance against inactivation because of freeze–thawing and freeze‐drying, were used as model proteins. The enzyme activities that remained after freeze–thawing and freeze‐drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze–thawing and freeze‐drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze‐drying than for the cryoprotection. The comprehensive comparison showed that the C10–C12 esters of sucrose or trehalose were the most effective through the freeze‐drying process: the remaining enzyme activities after freeze–thawing and freeze‐drying increased at the sugar ester concentrations of 1–10 and 10–100 μM, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein‐stabilizing effect of a small amount of sugar ester through the freeze‐drying process could be enhanced. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
Title compounds were synthesized on a preparative scale using alkaline phosphatase, orthophosphoric monoester phosphohydrolase B.C. 3.1.3.1, in reverse hydrolysis conditions. Optimization for one of the 25 phosphoryl acceptors investigated (glycerol) shows that up to 55% synthesis yield can be obtained using a large excess of substrate, conditions in which the enzymatic activity remains high. From the results obtained with different phosphoryl group donors, phosphate, pyrophosphate and polyphosphates and with enzymes of different sources, it comes up that the best results are obtained with pyrophosphate and with the weakly purified calf intestine alkaline phosphatase. The extent of enzymatic hydrolysis of the donor can be reduced owing to the existence of two different pH optima for the two reactions, phosphorylation and hydrolysis. The synthesis can be also performed using inert co-solvents which allow to reduce the amount of acceptor used, as long as Zn++ is added to the reaction medium. The results are discussed in terms of the catalytic mechanism of alkaline phosphatase.
1.
Bei den penicillinresistenten Staphylokokken wird eine Verminderung der oberflächlich verteilten Ribonucleinsäure festgestellt.
2.
Im Gehalt der von Trypsin angreifbaren Peptidverbindungen besteht bei den penicillinempfindlichen und penicillinresistenten Staphylokokken kein Unterschied.
3.
Die Staphylokokkenoberfläche ist arm an Monoester der Phosphorsäure.
1.1. In four species of cephalopods an investigation was made of the presence of several histochemically localizable enzymes.2.2. Among the enzymes studied were the cholinesterases, β-glucuronidase, various dehydrogenases, NADH2- and NADPH2-tetrazolium reductases, cytochrome c-oxidase, and several phosphatases.3.3. The main sites of hydrolysis of acetylcholine were the retinal plexus, some of the inner layers of the plexiform zone of the deep retina and the fibers of the optic ganglion. In decapods butyrylcholine was split at the same places, but in octopods the outer granular layer of the deep retina and the outer layers of the plexiform zone were the sole sites of hydrolysis in the optic lobe.4.4. β-glucoronidase was found in the distal segment of the retina and in the proximal segment in a zone just distal to the nuclei of the recepter cells. No other sites of activity could be demonstrated.5.5. NADH2-terazolium reductase occurred in all parts of the retina, in the optic nerves and in the optic lobe, the highest concentration being found in the plexiform zone. The same is true for the NAD-linked dehydrogenases, of which isocitrate dehydrogenase and α-cerophosphate dehydrogenase are the only enzymes to be found in high amounts.NADPH2-tetrazolium reductase occurred in the proximal segment of the retina, in the optic nerves and in the optic lobe, again in highest concentration in the plexiform zone. Succinate dehydrogenase shows the same localization as NADH2-tetrazolium reductase but occurs in essentially smaller amounts. Cytochrome c-oxidase is present in the same localization as NADH2-tetrazolium reductase.6.6. Alkaline and acid phosphatases were found in the retina and in all cells of the optic lobe. No activity could be detected in the nerve fibers or in the capillaries.A phosphatase splitting creatine phosphate at low pH, essentially different from acid phosphatase, has been demonstrated. Adenosine-monophosphatase and adenosinetriphosphatase occur in cells as well as axons of the optic system. The former enzyme is found in the highest concentration in the second radial layer of the plexiform zone, the latter is the only specific phosphatase found in the capillary walls.7.7. The main difference between the mammalian nervous system and the cephalopod optic ganglion were: the divergent behavior of true and aspecific cholinesterase towards specific inhibitors, the uniform moede of distribution of the reductases, dehydrogenases and cytochrome oxidase and the occurrence of alkaline phosphatase in the perikarya.
Optimum conditions for measuring the hydrolysis of disodium phenyl phosphate by alkaline phosphatase in raw milk included a 0.02919 M substrate concentration and a hydrolysis period of 20 minutes at 38.5° C. and pH 10.32 ± 0.02. These conditions differ from those specified in the Sanders and Sager method for determination of phosphatase activity in unheated milk.
Some properties of the enzyme, as it occurs in raw milk, were studied. The pH optimum of the catalyzed reaction increased with increasing concentrations of disodium phenyl phosphate. Where the substrate was sodium β-glycerophosphate, increased substrate concentration did not shift the pH optimum of 9.60 ± 0.02. Michaelis constants were 0.00340 M and 0.00560 M for disodium phenyl phosphate and sodium β-glycerophosphate, respectively. The energy of activation of the hydrolysis of disodium phenyl phosphate was approximately 9,000 cal / mole. In the absence of substrate, phosphatase was most stable at approximately pH 6.7. A slow decrease in the pH of raw milk resulted in less enzyme inactivation than a rapid decrease. Furthermore, the extent of subsequent restoration of phosphatase activity was greater at the normal pH of milk than at a higher pH. Added magnesium ion did not increase phosphatase activity under the experimental conditions.
The regularities of interactions of the main groups of substrates during membrane digestion, their mechanisms as well as physiological and possible clinical significance are characterized. Mono-, bi- and multisubstrate processes of the hydrolysis of proteins, fats, carbohydrates and phosphoric esters have been investigated using new experimental models. The authors show that the activity of enzymes ensuring membrane digestion is affected by various food substrates which do not serve as substrates for a given enzyme. The inhibitory and stimulating effects have been observed in the process of interactions between different groups of food substrates (for example, lipids and peptides) as well as within a single group of food substrates (for example, between different peptides, peptides and amino acids etc.). The existence of species, ontogenetic and organ characteristics of the processes of interactions at the stage of membrane hydrolysis is shown. The analysis of the mechanisms of interactions permits us to conclude that a number of enzymes accomplishing membrane digestion are allosterically regulated systems. The possible alterations in the regulatory properties of digestive enzymes in pathology are considered.
The different models proposed in the literature for the membrane of the milk fat globules are reviewed. On the basis of recent investigations, in particular of those of S. Hayashi, a new model is proposed, according to which the membrane is constituted by two layers which can easily be fractionated. The inner layer of cytoplasmic origin has a matrix structure, constituted by a complex lipid-protein aggregate. On this inner membrane an outer layer composed of numerous lipoprotein particles is associated. This new model will enable a better understanding of the alterations occurring in the fat globules phase during the various treatments in dairy chemistry. As an instance a tentative model is proposed for the copper migration during butter manufacture and the copper content of these butters.Moderne Auffassungen über die physikalische Struktur der Membran von Fettkügelchen in Milch und Sahne und die mögliche Beziehung der Struktur zur Wanderung des Kupfers während der Butter-HerstellungNach einer Literatur-Übersicht über die verschiedenen Strukturmodelle für die Membran von Milchfett-Kügelchen schlägt der Vf. unter Berücksichtigung neuerer Untersuchungen, insbesondere von S. Hayashi, ein neues Strukturmodell vor, demzufolge die Membran aus zwei Schichten besteht, die leicht fraktioniert werden können. Die innere Schicht, die aus Zytoplasmen stammt, besitzt eine Matrix-Struktur, die aus komplexen Lipoid-Eiweiß-Aggregaten besteht. Diese innere Membran ist mit einer aus zahlreichen Lipoprotein-Teilchen bestehenden äußeren Schicht verbunden. Das neue Modell ermöglicht ein besseres Verständnis der in den Fettkügelchen während der verschiedenen Behandlungen im Molkerei-Betrieb auftretenden Veränderungen. Als Beispiel werden mit Hilfe des Modells die Wanderung des Kupfers während der Butter-Herstellung sowie der Kupfer-Gehalt der Butter behandelt.
Cow's milk alkaline phosphatase. II. Subunit structure, metalloproteic nature and kinetic parametersAlkaline phosphatase (EC. 3.1.3.1) from cow's milk as a dimer comprising two identical or very similar subunits of about 85 000 molecular weight. The enzyme contains 4.9 ± 0.6 gatoms of zinc per mol of protein. The essential kinetic properties are the same as those of other alkaline phosphatases: variation of pH optimum value, the lack of specificity, increase of Km and V with pH value. The phosphotransferase activity is enlarged, at constant concentration of acceptor, with an increasing concentation of donor. The small size of molelcules and the presence of hydroxyls and amino groups increase the percentage of transfer phosphate. The phosphotransferase reaction is better with the d-isomer of serine and the enzyme possesses a more important affinity for the d-phosphoserine.RésuméLa phosphatase alcaline (EC 3.1.3.1) du lait de vache est un dimére comprenant deux sous-unités identiques ou trés similaires d'une masse moléculaire d'environ 85 000. L'enzyme contient 4.9 ± 0.6 atomes-g de zinc par mol de protéine. Les propriétés cinétiques essentielles sont celles des enzymes de son groupe: forte variation de la valeur du pH optimum avec les conditions d'essai, manque de spécificité de substrat, augmentation du Km et de V avec la valeur du pH. L'activité transférasique se trouve augmentée, à concentration fixe d'accepteur, en présence de concentration croissante de donneur. La faible taille des molécules et la présence de groupements hydroxyles et amine accroissent le pourcentage de phosphate transféré. La réaction de transphosphorylation se fait de préférence avec l'isomére d de la sérine et l'enzyme présente une plus grande affinité pour la D-phosphosérine.
Zusammenfassung Es wird eine neue Bestimmungs-methode für die Aktivität der alkalischen Phosphatase im Serum beschrieben, die Phospho-enol-pyruvat als Substrat benutzt. Durch Enzymwirkung freigesetztes Pyruvat wird zu Lactat hydriert und dieser Vorgang im Spektrophotometer verfolgt. Die Eigenschaften dieses optischen Tests werden eingehend diskutiert.
Chemiluminescent assays for beta-D-galactosidase and alkaline phosphatase are described which use novel chemiluminogenic substrates, o-aminophthalylhydrazide-beta-D-galactoside and o-aminophthalylhydrazide-O-phosphate, respectively. Each substrate is enzymatically hydrolyzed under the optimum conditions to release luminol, which luminesces in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The detection limits (S/N=3) of the enzyme activities (muU per tube) were 50 for beta-D-galactosidase and 100 for alkaline phosphatase.
1.1. Alkaline phosphatase activity was present in all tissues from all species examined.2.2. The tissue specific enzyme activities in organs from different species are within a comparable range when expressed in milliunits/mg protein.3.3. The enzymes from different species and organs have a definite and reproducible pattern of amino acid inhibition. The use of five amino acids (l-phe, l-arg, l-homoarg, l-met and l-val) enable us to establish this pattern in vitro and to identify in some cases the organ source.4.4. Results obtained from amino acid inhibition studies with alkaline phosphatases from one organ or species are valid only for that very organ in that very species.
The kinetics of the non-enzymatic hydrolysis of p-nitrophenyl phosphate have been studied in aqueous solution in the pH range 2.6 to 9.0 and at temperatures from 68.0°to 82.0 °C. The reaction has been followed by colorimetric measurement of the nitrophenol produced by the reactionThe reaction is first order with respect to p-nitrophenyl phosphate and has an activation energy of 26.0 kcal./mole at pH 2.6. An explanation has been proposed in terms of the different rates of hydrolysis of the various ionic species of the ester present in solution.
The phosphorus fraction made available to organisms by the action of alkaline phosphatase, here termed phosphatase hydrolyzable phosphorus (PHP), was studied in central New York State streams. Annual mean PHP concentrations were <2.0 μg P∙L−1 for each of the four streams studied and were low relative to concentrations for lentic systems from the literature. The PHP to soluble reactive phosphorus (SRP) ratio, a measure of the relative importance of PHP for aquatic organisms, was typically in the 0.09–1.00 range, indicating that PHP could supply a significant amount of P at certain locations. Laboratory sorption experiments with varying ionic strength solutions and with Ca2+ versus Na+ suggested that PHP was probably bound to sediments by a different mechanism than SRP, one probably mediated by the organic moiety. Fine-grained sediments higher in organic matter sorbed excess PHP from solution at a greater rate than coarse-grained sediments, but also released more into PHP-depleted solutions.
Based on the inhibition of double strand DNA (dsDNA) templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), a novel label-free turn-on fluorescent strategy to detect alkaline phosphatase (ALP) under physiological conditions has been developed. This method relies on the strong interaction between PPi and Cu2+, which would hamper the effective formation of fluorescent CuNPs, leading to low fluorescence intensity. The ALP-catalyzed PPi hydrolysis would disable the complexation between Cu2+ and PPi, facilitating the formation of fluorescent CuNPs through the reduction by ascorbate in the presence of dsDNA templates. Thus the fluorescence intensity was recovered and the fluorescence enhancement was related to the concentration of ALP. This method is cost-effective and convenient without any labels or complicated operations. The present strategy exhibits a high sensitivity and the turn-on mode provides a high selectivity for ALP assay. Additionally, the inhibition effect of phosphate on ALP activity was also studied. The proposed method using PPi substrate may hold a potential application in diagnosis of ALP-related diseases or evaluation of ALP functions in biological systems.
This chapter discusses the molecular and catalytics properties of mammalian alkaline phosphatases. Alkaline phosphatases catalyze the hydrolysis of almost any phosphomonoester to give P1 and the corresponding alcohol, phenol, or sugar, etc. They are distinguished from a similar group of enzymes, the acid phosphatases, primarily by the pH dependence of their catalytic activity, which is higher in the alkaline range than at neutral or acid pH. Studies have shown that (alkaline) phosphatases extracted from many mammalian tissues, particularly kidney and intestinal mucosa, could hydrolyze nucleotides, hexosephosphates, and glycerophosphate. Robison's demonstration that ossifying cartilage was a rich source of phosphatase marked the beginning of a phase of intensive investigation, developing the hypothesis that phosphatase was directly involved in the process of calcification. Bone phosphatase was found to have an optimum activity at pH 8.4-9.4 similar in this respect to the enzymes from kidney and intestinal mucosa and to that present in blood plasma. Other tissues with relatively high phosphatase activity, such as liver and spleen, were shown to contain an additional enzyme with optimum activity a t pH 5. Davies proposed they be termed alkaline and acid phosphatase, respectively, by which names they are currently accepted. Erdtman made the important observation that kidney phosphatase, after dialysis, required Mg2+ for maximum activity. This was confirmed and extended to include all mammalian alkaline phosphatases whether dialyzed or not and has led to the general practice of including Mg2+ in the assay medium.
1.1. Various substrates were injected daily into chick eggs to test their inductive effect on phosphatases in the duodenum, liver, mesonephros, and metanephros of embryos between 14 and 19 days of incubation.2.2. Only phenyl phosphate administration significantly increased the activity of alkaline phosphomonoesterase in all organs studied. None of the substrates affected acid phosphomonoesterase activity. Adenosine-5-phosphatase activity was not affected by phenyl phosphate administration.3.3. Various observations indicate that the observed increase in alkaline phosphomonoesterase activity is to be taken as an actual synthesis of the enzyme.4.4. The patterns of alkaline phosphomonoesterase accumulation following phenyl phosphate administration were analyzed in detail.5.5. Histological and histochemical observations show that the induced alkaline phosphomonoesterase has an identical localization with the normal enzyme, and that the induced synthesis of the enzyme is dissociable from morphological differentiation of the organs to a considerable extent.6.6. The results obtained were discussed on the basis of the properties and suggested functions of phosphatases from an embryological viewpoint.
IntroductionEarly Zinc(II)-Macrocyclic Polyamine Complexes Relevant to the Present StudyVarious Acid Properties of Zinc(II) in Macrocyclic Polyamine ComplexesStructural Similarities of the [12]aneN3-ZnII-OH- with the Active Center of Zinc(II) in Carbonic AnhydraseStrong Anion Affinities of ZnII-[12]aneN3 AND carbonic AnhydraseThe Action of ZnII-OH- as a NucleophileZnII-OH- as a Base Toward Neutral Aromatic SulfonamidesA Carbonic Anhydrase-Substrate HCO3 Affinity for Zinc(II)The ZnII-OH- as a Base Towards CarboxyamidesThe [12]aneN3-ZnII-OH- Species as a New Alcohol Dehydrogenase ModelConcluding Remarks on the Intrinsic Acid Properties of Zinc(II) for Hydrolytic Zinc Enzymes
1.1. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase EC 3.1.3.1.) was extracted from the small intestines of the rainbow lizard Agama agama, partially purified by DEAE-cellulose and Sephadex G-200 column chromatography and characterized.2.2. The enzyme had an optimum pH at 9.5 in sodium carbonate/bicarbonate buffer: a Km of 1.6 mM with p-nitrophenyl phosphate; a molecular weight of 132,000; was inhibited by Zn2+, EDTA, urea and phenylalanine; stimulated by Co2+, Mn2+ and Mg2+, but Ca2+ had little or no effect on the activity of the enzyme.3.3. The inhibition by urea was non-competitive, that by phenylalanine was uncompetitive. The enzyme was heat-labile.
1.
In einem Ftterungsversuch wurde der Mangangehalt der Milch nur geringfgig durch Zustze von MnSO4 zum Futter beeinflut.
2.
Zwischen dem Mangangehalt der Milch und der Aktivitt der alkalischen Milchphosphatase besteht ein statistisch gesicherter Zusammenhang.
1.1. The alkaline phosphatase activity of the small intestine of the mature mouse is highest at the pyloric end of the duodenum and falls sharply to a much lower level in the jejunum.2.2. The high level of activity in the duodenum is accompanied by a high preference for phenylphosphate (PhP) over β-glycerophosphate (bGP). Jejunal and ileal phosphatases show a preference for bGP at maximal activity levels.3.3. In the preweaning period alkaline phosphatase activity is uniform throughout the small intestine. All areas hydrolyze bGP more effectively than PhP at this time.4.4. The great increase in alkaline phosphatase activity that occurs in the duodenum during the third postnatal week (i.e., just before weaning) involves the appearance of a strong preference for PhP. Further kinetic studies suggest that the preweaning increase in activity in the duodenum represents the synthesis of a new enzyme not previously present in significant quantity.5.5. During the third postnatal week jejunal and ileal phosphatase activities rise and fall. The changes are very small in comparison with the large increase occurring concomitantly in the duodenum.6.6. In the duodenum, exogenous cortisone brings about the precocious development of large quantities of a phosphatase having a decided preference for PhP.7.7. In the jejunum, exogenous cortisone has no influence on phosphatase activity. In the ileum it first depresses, then elevates, phosphatase activity, but the induced alterations do not parallel the normal pattern of change in the preweaning period.8.8. Histochemical studies show that the most active phosphatases are localized in the microvilli throughout the intestine. Surface measurements show that differences in activity cannot be attributed to differences in amount of microvilli-bearing surface along the length of the intestine.
The development and application of a method for the determination of pesticides and heavy metals by means of a biosensor system are described. Chemometrics, especially experimental design and simplex optimization proved to be a very valuable tool for this task. The system consists of three pH electrodes whose ion sensitive areas are connected with a membrane on which one enzyme is immobilized at a time. As biological components, acetylcholinesterase, alkaline and acid phosphatase were used; the latter two were employed for the first time in such a way. By potentiometrically detecting the pH shifts of the typical reactions of the immobilized enzymes with the corresponding substrates before and after an incubation time, the relative inhibition of eleven pesticides was determined by forming the ratio of both the potential differences. The combination of the inhibition results of three enzyme electrodes enables to get more information, both additional and excluding, about these pollutants than with only one enzyme electrode. In this way, a separation scheme of the used substances to be detected could be derived.
The alkaline phosphatase of human placenta, prepared and purified as described in previous papers1,2 hydrolyses β-glycerophosphate and phenyl at phosphate at nearly the same rate. The alkaline phosphatases of the kidney and the intestinal mucosa, on the other hand, hydrolyze phenyl phosphate much more rapidly than β-glocero-phosphate. The Michaelis-Menton constant, Km, for placental alkaline phosphatase is also different.
The alkaline phosphatase of bovine milk has been purified more than 1000-fold by dissociation of the phosphatase complex with n-butanol and subsequent fractionation with acetone. This extends the usefulness of the n-butanol treatment originally used for the purification of the alkaline phosphatase in cream.
IN general, enzymes concerned with the transfer of 'high-energy' phosphate depend for their activity on the presence of free thiol groups, and it has been suggested that the enzyme-substrate combinations of the phosphokinases may involve the formation of - SH - Mg - phosphate complexes1. The phosphatases, adenosine-triphosphatase2 and hexose-diphosphatase3, have also been shown to be sulphydryl enzymes.
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