Placental alkaline phosphatase determination by inhibition with ethylendiaminetetracetic acid

Miggiano, GA; Pileri, M; Mordente, Alvaro; Martorana, GE; Castelli, A

doi:10.1016/0009-8981(85)90042-7

Cited by 6 publications

(5 citation statements)

References 13 publications

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“…Selective inhibition employing EDTA was also studied in 40 mmol/1 AMP buffer, pH 10.5, with a 10 mmol/1 final concentra tion of EDTA, as previously suggested [16]. The residual activity was reduced to about 65% when the sample was preincubated for 5 min at 37 °C.…”

Section: Resultsmentioning

confidence: 99%

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Alkaline Phosphatase Expression in Human Chorionic Villi

et al. 1987

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The physicochemical properties and electrophoretic mobility of different isoforms of alkaline phosphatase were studied in chorionic villi. Based on selective inactivation and inhibition studies (thermal stability, inactivation by urea, EDTA and L(+)ascorbic acid and L-amino acid inhibition), evidence was obtained for the existence of two distinct types of alkaline phosphatase in trophoblast cells. One type is peculiar to chorionic villi while the other is also found in term placenta. Both show two isoforms. These two isoforms were observed with polyacrylamide gel electrophoresis, carried out at pH 6.0 and 9.5. It is suggested that the qualitative and quantitative methods of alkaline phosphatase analysis could be used for first trimester fetal diagnosis of severe infantile hypophosphatasia and for understanding genetic control during early fetal development.

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Section: Resultsmentioning

confidence: 99%

“…EDTA inhibition studies were performed at fixed concentrations (10 mmol/1 EDTA in 40 mmol/1 AMP buffer solution, pH 10.5) with a preincubation time ranging between 0 and 5 min, as previously described for placental A1P inhibition [16].…”

Section: L-aminomentioning

confidence: 99%

Alkaline Phosphatase Expression in Human Chorionic Villi

et al. 1987

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“…The kinetic behaviour of the intestinal form diverges from that of other AP isoenzymes upon the addition to the reaction mixture of different modifiers such as urea (Miggiano et al, 1984) and EDTA (Conyers et al, 1967;Miggiano et al, 1985a).…”

Section: Introductionmentioning

confidence: 97%

Early conformational changes and activity modulation induced by guanidinium chloride on intestinal alkaline phosphatase

Miggiano

Mordente

Pischiutta

et al. 1987

Biochemical Journal

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Moderate concentrations of guanidinium chloride induce both instantaneous and time-dependent modifications of the catalytic and optical properties of intestinal alkaline phosphatase, which undergoes consecutive conformational transitions at about 0.05 M, 0.25 M and 1.0 M denaturant. A paradoxical activation is observed up to 1.0 M-guanidine, with a maximum at 0.25 M- and a mid-point around 0.5 M-guanidine. Difference absorbance and fluorescence spectra imply a change in the state of ionization of the protein residues, with variation in molecular size suggested by light-scattering. Random-coil formation is indicated by a lower fluorescence yield, a more polar environment of the aromatic residues and another separate tryptophan emission. Iodide quenching confirms the alterations of conformation. Deprotonation favours the loss of the intramolecular constraints and the enhancement of the structure disruption by guanidine.

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“…The inactivation of mammalian alkaline phosphatases by chelating agents is well documented (Ackerman & Ahlers, 1976), and that by EDTA has been examined by several groups (Conyers et al, 1967;Ackermann & Ahlers, 1976;Miggiano et al, 1985). From these studies it is evident that the effects of EDTA are not entirely straightforward in that it causes an immediate inhibition as well as a time-dependent inactivation (Conyers et al, 1967).…”

Section: Discussionmentioning

confidence: 99%

Resistance to inactivation by EGTA of the enzyme-substrate and enzyme-phosphate complexes of alkaline phosphatase

Pike

Duggleby

1987

Biochemical Journal

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Bovine intestinal mucosal alkaline phosphatase is inactivated by the chelating agent EGTA. Several concentrations of the enzyme were incubated with EGTA and a range of concentrations of the substrate p-nitrophenyl phosphate to determine the substrate concentration as a function of time. As predicted by a recently developed theory [Duggleby (1986) J. Theor. Biol. 123, 67-80], catalysis ceases before all substrate is exhausted. An analysis of these final substrate concentrations according to the theory revealed that, whereas the free enzyme is unstable, the effect of EGTA is counteracted when either the substrate or product (phosphate) is bound. Comparison of the results with those obtained by direct stability measurements and steady-state kinetic experiments gave a qualitatively and quantitatively consistent body of evidence in support of this interpretation.

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Placental alkaline phosphatase determination by inhibition with ethylendiaminetetracetic acid

Cited by 6 publications

References 13 publications

Alkaline Phosphatase Expression in Human Chorionic Villi

Alkaline Phosphatase Expression in Human Chorionic Villi

Early conformational changes and activity modulation induced by guanidinium chloride on intestinal alkaline phosphatase

Resistance to inactivation by EGTA of the enzyme-substrate and enzyme-phosphate complexes of alkaline phosphatase

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