Kinetic determination of alkaline phosphatase activity based on hydrolytic cleavage of the PF bond in monofluorophosphate and fluoride ion-selective electrode

Venetz, Werner; Mangan, Ciaran; Siddiqi, I.

doi:10.1016/0003-2697(90)90398-s

Cited by 15 publications

(7 citation statements)

References 13 publications

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“…The assay utilizes an alternative substrate, (2 S ,4 S )-4-fluoroproline (flp), that upon turnover by P4H forms (2 S )-4-ketoproline (Kep) and releases inorganic fluoride [30]. The rate of substrate turnover is monitored continuously by using a fluoride ion-selective electrode, which has been employed previously in assays for alkaline phosphatase and chondroitin AC lyase [31,32]. Our assay for P4H yields kinetic parameters comparable to those from a discontinuous HPLC-based assay using the same peptide substrate.…”

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confidence: 99%

Direct and continuous assay for prolyl 4-hydroxylase

Gorres

Raines

2009

Analytical Biochemistry

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Prolyl 4-hydroxylase (P4H) is a non-heme iron dioxygenase that catalyzes the post-translational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O 2 as co-substrates, and forms succinate and CO 2 as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLCbased assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.Collagens are the major structural proteins of the extracellular matrix. Collagens undergo a number of post-translational modifications during biosynthesis. One major modification is the hydroxylation of certain prolyl residues. The repeating amino-acid sequence of collagen, wherein every third residue is a glycine (Gly): Xaa-Yaa-Gly, is rich in (2S)-proline (Pro). Indeed, Pro is the most common amino acid found in the Xaa position [1]. In the Yaa position, the most common amino is (2S, 4R)-4-hydroxyproline (Hyp). Hyp is formed by a posttranslational modification of Pro catalyzed by prolyl 4-hydroxylase (P4H; EC 1.14.11.2).Hyp is necessary for the stable formation of the triple-helical structure of collagen. Collagen with decreased levels of Hyp is defective in folding and secretion under physiological conditions [2][3][4]. P4H activity is required for the viability of the nematode Caenorhabditis elegans [5,6] and the mouse Mus musculus [7]. In vitro, P4H has been studied by availing enzyme via heterologous expression in insect cells, yeast, and (only recently [8,9]) bacteria. *Corresponding author. Tel: 608-262-8588. Fax: 608-890-2583. E-mail address: rtraines@wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the conten...

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confidence: 99%

Direct and continuous assay for prolyl 4-hydroxylase

Gorres

Raines

2009

Analytical Biochemistry

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“…It is believed that monofluorophosphate would exert its caries-preventive efficacy after the splitting of the molecule to free fluoride (F -) and phosphate ions by phosphatase enzymes in the oral cavity (26,27). In this study, no such enzyme was used because of several difficulties in use of enzymes, i.e., no commercially available and validated enzyme identical to phosphatase enzymes in the oral cavity, no information on how to use them in active conditions using a standardized procedure without interfering with dentifrice ingredients such as sodium laurylsulfate, and others (28).…”

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confidence: 99%

Effect of a test dentifrice containing nano-sized calcium carbonate on remineralization of enamel lesions in vitro

et al. 2009

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The efficacy of a test dentifrice containing nano-sized (several tens to hundreds of nm) calcium carbonate (hereafter NC) on enamel lesion remineralization was studied in an in vitro system that employed collagen-coated wells for cell culture, as a model of oral surfaces for NC retention. The well surfaces were treated with the test dentifrice and briefly rinsed with distilled water. Thin sections of enamel with artificial subsurface demineralization were remineralized in the plate wells containing remineralizing solution. The dentifrice treatment was repeated twice a day (in the morning and evening) for 20 days. After remineralization, microradiographic analysis was performed to evaluate the rate of lesion remineralization on the sections. The test dentifrice showed a statistically significant mineral gain (48.8% decrease in DeltaZ % x microm from the baseline value), indicating lesion remineralization, whereas the placebo dentifrice without NC did not. An elevated Ca concentration in the remineralizing solution was also observed after a single treatment with the test dentifrice. We conclude that the test dentifrice has potential to remineralize incipient enamel lesions due to the unique properties of NC, which is retained on oral surfaces, thereafter releasing Ca ions into oral fluids (saliva, plaque).

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“…Η θερμοκρασία αναμένονταν, σύμφωνα με τα δεδομένα της βιβλιογραφίας [40], να είναι μια κρίσιμη παράμετρος της ενζυμικής αντίδρασης. Παρόλα αυτά, τα πειραματικά αποτελέσματα έδειξαν μη σημαντική επίδραση της θερμοκρασίας στη δραστικότητα του ακινητοποιημένου ενζύμου.…”

Section: μελέτη των παραμέτρων της ενζυμικής αντίδρασηςunclassified

“…Η επόμενη παράμετρος που μελετήθηκε ήταν η επίδραση της συγκέντρωσης ποσότητας του ιόντος Mg(II) στο δείγμα, στην ενζυμική αντίδραση. Από τη βιβλιογραφία είναι γνωστό ότι το ιόν Mg(II) δρα ως ενεργοποιητής σε αντιδράσεις της AlPase [40,41]. Όπως φαίνεται και στο σχήμα 10.9, υπάρχει συμφωνία μεταξύ των πειραματικών αποτελεσμάτων και της βιβλιογραφίας.…”

Section: μελέτη των παραμέτρων της ενζυμικής αντίδρασηςunclassified

“…Πρόσφατες έρευνες απέδειξαν ότι το ιόν MFP "αναγνωρίζεται" ως υπόστρωμα από το ένζυμο αλκαλική φωσφατάση (AlPase) [40,41]. Με βάση τις έρευνες αυτές, το ιόν MFP χρησιμοποιήθηκε ως υπόστρωμα, με σκοπό τον υπολογισμό της ενεργότητας του ενζύμου, εφαρμόζοντας ποτενσιομετρική ανίχνευση (εκλεκτικό ηλεκτρόδιο ιόντος F -) [40] και ως ένωση-μοντέλο, με σκοπό τη μελέτη της ενζυμικής διάσπασης του τοξικού αερίου Sarin [41]. Οι παραπάνω μελέτες απέδειξαν ότι παρόλο που το ιόν MFP δεν υπάρχει στη φύση, "αναγνωρίζεται" από την AlPase ως υπόστρωμα και πως το ένζυμο καταλύει την υδρόλυση του δεσμού P-F.…”

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Ανάπτυξη μεθόδων προσδιορισμού ορισμένων ανιόντων με την τεχνική της έγχυσης του δείγματος σε συνεχή ροή - fia

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Καθηγητής Ιωάννης Παπαδογιάννης -(Μέλος συμβουλευτικής επιτροπής) Καθηγητής Ιωάννης Στράτης -(Μέλος συμβουλευτικής επιτροπής) Καθηγητής Αναστάσιος Βουλγαρόπουλος -(Αριστοτέλειο Πανεπιστήμιο Θεσ/νίκης) Καθηγητής Αντώνιος Καλοκαιρινός -(Καποδιστριακό Πανεπιστήμιο Αθηνών) Καθηγητής Μιχαήλ Κουππάρης -(Καποδιστριακό Πανεπιστήμιο Αθηνών) Λέκτορας Αναστάσιος Οικονόμου -(Αριστοτέλειο Πανεπιστήμιο Θεσ/νίκης) Περιεχόμενα i ΠΕΡΙΕΧΟΜΕΝΑ Πρόλογος Εισαγωγή ΘΕΩΡΗΤΙΚΟ ΜΕΡΟΣ 1. Αυτοματοποιημένοι αναλυτές με έγχυση του δείγματος σε συνεχή ροή -FIA

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Kinetic determination of alkaline phosphatase activity based on hydrolytic cleavage of the PF bond in monofluorophosphate and fluoride ion-selective electrode

Cited by 15 publications

References 13 publications

Direct and continuous assay for prolyl 4-hydroxylase

Direct and continuous assay for prolyl 4-hydroxylase

Effect of a test dentifrice containing nano-sized calcium carbonate on remineralization of enamel lesions in vitro

Ανάπτυξη μεθόδων προσδιορισμού ορισμένων ανιόντων με την τεχνική της έγχυσης του δείγματος σε συνεχή ροή - fia

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