scholarly journals Potent inhibition of membrane-bound rat intestinal alkaline phosphatase by a new series of phosphate analogues

1981 ◽  
Vol 194 (3) ◽  
pp. 797-802 ◽  
Author(s):  
S P Shirazi ◽  
R B Beechey ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Intestinal Alkaline Phosphatase ◽  
Irreversible Inhibitor ◽  
Intestinal Brush Border Membrane ◽  
Potent Inhibition ◽  
Competitive Inhibitors ◽  
Membrane Bound ◽  
Ribose Moiety

The inhibition by phosphonates and phosphate analogues of the alkaline phosphatase activity of rat intestinal brush-border membrane vesicles was studied at pH 7.5 and 30 degrees C. Phenylene-1,3-diphosphonate, 2,6-dinitrophenylphosphonate and phosphonoacetaldehyde were found to be competitive inhibitors, with Ki values in the range 16-80 microM. Adenosine 5′-[beta-thio]diphosphate and adenosine 5′[gamma-thio]triphosphate are also very potent inhibitors, with Ki values of approx. 10 microM. The inhibition produced by these thiophosphates was mainly competitive but with a slight non-competitive element. Adenosine 5′-[beta gamma-imido]triphosphate is also a competitive inhibitor of the alkaline phosphatase, but oxidation of the ribose moiety of this compound with NaIO4 results in an active-site-directed irreversible inhibitor that could be of general use in studies of the mechanism of action of this enzyme.

Na-dependent L-glutamate transport by eel intestinal BBMV: role of K+ and Cl-

1989 ◽  
Vol 257 (1) ◽  
pp. R180-R188
Author(s):  
P. M. Romano ◽  
G. A. Ahearn ◽  
C. Storelli
Keyword(s):  
Amino Acid ◽  
Glutamate Uptake ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Anguilla Anguilla ◽  
Competitive Inhibitor ◽  
Glutamate Transport ◽  
Electrical Potential Difference ◽  
Intestinal Brush Border Membrane ◽  
Transmembrane Electrical Potential

L-[3H]glutamate uptake into eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) was a sigmoidal function of extravesicular Na, suggesting that two or more cations accompanied the amino acid during transport. L-[3H]glutamate influx illustrated the following kinetic constants: apparent membrane binding affinity (Kapp) = 0.80 +/- 0.12 mM; influx velocity (Jmax) = 2.61 +/- 0.31 nmol.mg protein-1.min-1; and permeability coefficient (P) = 0.65 +/- 0.10 microliters.mg protein-1. min-1. Results from the imposition of diffusion potentials across vesicle membranes using K-valinomycin or H-carbonyl-cyanide p-chloromethoxyphenylhydrazone suggested that Na-dependent L-glutamate transport was sensitive to transmembrane electrical potential difference. Extravesicular aspartate was a competitive inhibitor of L-[3H]glutamate influx [inhibitory constant (Ki) = 0.28 +/- 0.04 mM]. Intravesicular K and extravesicular Cl ions enhanced maximal amino acid influx and transient L-glutamate accumulation against a concentration gradient (overshoot). Intravesicular K reduced the Kapp of the membrane to L-glutamate, whereas extravesicular Cl increased L-glutamate Jmax. A model for L-[3H]glutamate transport is suggested involving the cotransport of at least two Na and one L-glutamate that is activated by one intravesicular K ion and at least two extravesicular Cl ions.

scholarly journals Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations

1967 ◽  
Vol 102 (3) ◽  
pp. 917-921 ◽  
Author(s):  
R.H. Eaton ◽  
D. W. Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Competitive Inhibitor ◽  
Tissue Preparation ◽  
Intestinal Alkaline Phosphatase ◽  
Active Centre ◽  
Low Concentrations ◽  
Type 3 ◽  
Intestinal Preparation ◽  
Hydrolysis Of ◽  
Single Enzyme

1. Inhibition of the pyrophosphatase and orthophosphatase activities of human liver and small-intestinal alkaline-phosphatase preparations by different classes of inhibitors has been studied. 2. Each type of substrate, pyrophosphate or orthophosphate, is a competitive inhibitor of hydrolysis of the other type. 3. l-Phenylalanine is a non-competitive inhibitor of both types of activity of the intestinal preparation, but inhibits neither activity of the liver enzyme. Arsenate is a competitive inhibitor of both activities of both preparations. For a given inhibitor, the values of K(i) are independent of the type of substrate used when measurements are made at the same pH. 4. Mg(2+) ions activate orthophosphatase but inhibit pyrophosphatase, except in very low concentrations. 5. These results are compatible with the presence in each tissue preparation of a single enzyme with one type of active centre, possessing both orthophosphatase and pyrophosphatase activities.

scholarly journals The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1969 ◽  
Vol 112 (5) ◽  
pp. 699-701 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Competitive Inhibitor ◽  
Inorganic Pyrophosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Low Concentrations ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of ◽  
Pyrophosphate Hydrolysis

1. The differential effects of adding Zn2+ and Mg2+ on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn2+, inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn2+–pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg2+ also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.

Differential Release of Human Intestinal Alkaline Phosphatase in Duodenal Fluid and Serum

1992 ◽  
Vol 38 (12) ◽  
pp. 2532-2538 ◽  
Author(s):  
J T Deng ◽  
M F Hoylaerts ◽  
V O Van Hoof ◽  
M E De Broe
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase D ◽  
Protein Complexes ◽  
Membrane Vesicles ◽  
Reference Population ◽  
Intestinal Alkaline Phosphatase ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Duodenal Fluid ◽  
Vascular Bed

Abstract Human intestinal alkaline phosphatase (IAP) can be released by the enterocyte into duodenal fluid as a mixture of three isoforms. A proportion of the enzyme is associated with triple-layered membrane vesicles (vesicular IAP). Although, occasionally, free hydrophilic IAP dimers are present, the remaining enzyme usually consists of a mixture of hydrophobic IAP dimers and more complex hydrophobic IAP structures of larger size, both entities being identified as "intestinal variant" alkaline phosphatase (VAR IAP). The hydrophobicity of VAR IAP stems exclusively from its attached glycosyl-phosphatidylinositol (GPI) anchor. Both vesicular IAP and VAR IAP are converted to hydrophilic enzyme upon removal of the GPI tail by phospholipase D (PLD) present in duodenal fluid. The IAP released into the vascular bed consists mainly of VAR IAP; vesicular IAP is absent. The enzyme characteristics of VAR IAP partially purified from duodenal fluid and from serum are identical. In plasma, VAR IAP appears to associate with (lipo)protein complexes and is thus protected from further degradation by plasma PLD. Such complex formation may explain why, in the serum of a healthy reference population, VAR IAP was more abundant than hydrophilic dimeric IAP.

scholarly journals Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

1981 ◽  
Vol 200 (3) ◽  
pp. 645-654 ◽  
Author(s):  
Graeme P. Young ◽  
Steven T. Yedlin ◽  
David H. Alpers
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Double Diffusion ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Intestinal Alkaline Phosphatase ◽  
Absolute Increase ◽  
Membrane Bound

Enzymically active intestinal alkaline phosphatase exists in both soluble and membrane-bound forms in the suckling rat. Antiserum prepared against purified soluble alkaline phosphatase (anti-AlP) was shown to be monospecific when assessed by Ouchterlony double-diffusion analysis and immunoelectrophoresis. The two forms of alkaline phosphatase were antigenically identical and possessed similar affinities for anti-AlP. To study the biosynthesis of the two forms, 14-day-old rats were injected intraperitoneally with [3H]leucine. The labelling kinetics of alkaline phosphatase, extracted from supernatant and brush-border membrane fractions with anti-AlP, was followed over 20h. Incorporation of [3H]leucine into membrane-bound alkaline phosphatase was rapid, reaching a plateau at 6h. The soluble enzyme showed slower incorporation of label and maximal radioactivity was not reached until 12h after labelling, a lag of 6h behind the membrane-bound enzyme. Soluble alkaline phosphatase could not have been a precursor of the membrane form, as there was no early peak of radioactivity in the soluble form. To determine if the soluble enzyme was irreversibly derived from the membrane enzyme, a newly developed technique of labelling brush-border membrane proteins in vivo by intraluminal injection of diazotized [125I]iodosulphanilic acid was used. The appearance of 125I in soluble and membrane alkaline phosphatase was then monitored over a 7h period, encompassing the lag between maximal leucine labelling of the two forms. The results failed to show either a proportional transfer of radioactivity from membrane to soluble alkaline phosphatase or an absolute increase in radioactivity of the soluble form during degradation of brush-border alkaline phosphatase. Therefore there does not appear to be a serial precursor/product relationship between the soluble and membrane-bound forms of suckling-rat intestinal alkaline phosphatase.

scholarly journals Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony

Toxins ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 409 ◽  
Author(s):  
Daniel Pinos ◽  
Maissa Chakroun ◽  
Anabel Millán-Leiva ◽  
Juan Luis Jurat-Fuentes ◽  
Denis J. Wright ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Heliothis Virescens ◽  
Insect Pests ◽  
Resistance Management ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Insect Midgut ◽  
Evolution Of Resistance ◽  
Membrane Bound ◽  
Mechanistic Basis

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

scholarly journals Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens

2020 ◽  
Author(s):  
Daniel Pinos ◽  
Maissa Chakroun ◽  
Anabel Millán-Leiva ◽  
Juan Luis Jurat-Fuentes ◽  
Denis J. Wright ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Heliothis Virescens ◽  
Insect Pests ◽  
Resistance Management ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Insecticidal Protein ◽  
Down Regulation ◽  
Cry Proteins ◽  
Membrane Bound

ABSTRACTThe Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests, thus understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, a laboratory-selected colony of Heliothis virescens (Vip-Sel) highly resistant to the Vip3Aa protein was used to test whether an alteration of membrane receptors in the insect midgut might explain the resistance phenotype. Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae show dramatically reduced levels of alkaline phosphatase activity, which was further confirmed by a strong down-regulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, its involvement as a receptor for the Vip3Aa protein was not supported by ligand blotting and viability assays with insect cells expressing HvmALP1. These data support that reduced alkaline phosphatase, previously observed in insect colonies resistant to Cry proteins from Bt, may also serve as an indirect marker that is not mechanistically involved in resistance to Vip3Aa.IMPORTANCEThe Vip3Aa insecticidal protein remains the only lepidopteran-specific trait in transgenic Bt crops with no cases of field-evolved resistance. While laboratory-selected resistance to Vip3A has been reported elsewhere, the mechanism for resistance is unknown. Results in this work show lack of significant Vip3Aa binding alterations in resistant and reference colonies of H. virescens. These observations are in contrast to most cases of high levels of resistance to insecticidal Bt proteins for which decreased binding is commonly detected. In addition, this study provides the first evidence of down-regulation of membrane bound alkaline phosphatase (mALP) associated with Vip3Aa resistance, a phenomenon commonly associated with resistance to Cry proteins from Bt. Results from this work suggest that mALP down-regulation may be a useful biomarker yet reject its direct participation in resistance to Vip3Aa.

Variant in C-terminal region of intestinal alkaline phosphatase associated with benign familial hyperphosphatasaemia

2018 ◽  
Vol 55 (10) ◽  
pp. 701-704 ◽  
Author(s):  
Takayuki Ishige ◽  
Sakae Itoga ◽  
Emi Utsuno ◽  
Motoi Nishimura ◽  
Masaharu Yoshikawa ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mechanism ◽  
Massively Parallel Sequencing ◽  
Genetic Diagnosis ◽  
Wild Type ◽  
Intestinal Alkaline Phosphatase ◽  
Membrane Bound ◽  
Long Range Pcr ◽  
In Vitro Protein Expression

BackgroundA genetic diagnosis has been rarely performed in benign familial hyperphosphatasaemia, and molecular mechanism largely remains unclear.ObjectivesWe encountered a case with benign familial hyperphosphatasaemia of intestinal alkaline phosphatase (IAP). To elucidate the molecular mechanism, we performed ALPI gene sequencing and in vitro protein expression analysis.MethodsALPI gene was sequenced by long-range PCR and massively parallel sequencing. The soluble and membrane-bound ALP activities of the cultured cell line, transfected with the wild-type or variant-type ALPI gene were analysed by a glycosylphosphatidylinositol (GPI)-cleaving assay.ResultsWe identified a deletion–insertion variant in the C-terminal end of the ALPI gene. This variant causes the attenuation of the hydrophobicity in GPI-anchor signal of IAP. An in vitro GPI-cleaving assay demonstrated that the membrane-bound IAP was greatly decreased, whereas the soluble IAP was increased, in the variant IAP.ConclusionsThe C-terminal variant in ALPI causes the benign familial hyperphosphatasaemia of IAP by the attenuation of the membrane-binding capability.

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