Kinetic behaviour of calf-intestinal alkaline phosphatase with 4-methylumbelliferyl phosphate

Fernley, H. N.; Walker, P. G.

doi:10.1042/bj0970095

Cited by 180 publications

(68 citation statements)

References 15 publications

(13 reference statements)

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“…Identification of dephosphorylation as the slow step would be in accord with the conclusions drawn by Fossett et al [7]. However, Fernley and Walker [8,10] interpreted the catalysed hydrolysis of 4-methylumbelliferyl phosphate monoester as slow phosphorylation (k2) rather than dephosphorylation above pH 6.0; and in order to explain the observed relationship between K,,, and Vand the pH dependences of their values, they showed a requirement for a mechanism involving two conformational forms of the enzyme related by a pH-dependent equilibrium.…”

Section: Discuss I 0 Nsupporting

confidence: 73%

“…The value is close to that reported by Fossett et al [7] for a catalytic group in free calf intestinal alkaline phosphatase, which, they concluded, must be in the unprotonated form for complete activity. Fernley and Walker [8] found that formation of an unprotonated complex characterised by a similar pK value increased the rate of hydrolyses catalysed by alkaline phosphatase (from the same source) by approximately sixfold. The identity of these groups were not eluciated however.…”

Section: Discuss I 0 Nmentioning

confidence: 99%

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Buffer-Induced Activation of Calf Intestinal Alkaline Phosphate

Bannister

Foster

2005

European Journal of Biochemistry

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The influence of Tris, phosphate ions, imidazole, carbonate ions, bicarbonate ions and hydroxyl ions on the rate of activation of calf intestinal alkaline phosphatase after its dissolution in aqueous solution was investigated. The kinetic data are consistent with a general base-catalysed reaction in which the base reacts with an unprotonated enzyme species formed by ionization of a group with pK = 8.9.Mammalian alkaline phosphatases are zinc metallo enzymes catalysing the hydrolysis of phosphomonoesters, and the transfer ofphosphoryl groups to suitable aminoalcohol acceptors. It has long been recognised that dilution of serum, with buffer, has resulted in an activation of alkaline phosphatase [l]. Similarly, reconstitution of lyophilised serum or thawing of frozen serum results in a slow activation of alkaline phosphatase [2,3]. However, this phenomenon has not been satisfactorily characterised. It has been attributed variously to dissociation of partially active lipoprotein complexes [4], reversible association of the enzyme with Tris to form a more active addition complex [5], or from calculations of entropy changes to exposure of additional active sites [6].This paper demonstrates that rapid dissolution of partially purified calf intestinal alkaline phosphatase into buffered solution is followed by an increase in activity, the rate of which depends on pH and buffer concentration. The attendant kinetics of activation are consistent with general base catalysis in which the conjugate base of the buffer activates the enzyme by reaction with an unprotonated species formed by ionisation of a group with pK = 8.9.

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Section: Discuss I 0 Nsupporting

confidence: 73%

Section: Discuss I 0 Nmentioning

confidence: 99%

Buffer-Induced Activation of Calf Intestinal Alkaline Phosphate

Bannister

Foster

2005

European Journal of Biochemistry

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“…We used a modification (Freiburghaus, Hauri, Green & Hadorn, 1978) of the method of Schmitz, Preiser, Maestracci, Ghosh, Corda & Crane (1973). Activities of aminopeptidase N (modified from Saifuku, Sekine, Namihisa, Takahashi & Kanaoka, 1978), dipeptidyl aminopeptidase IV (Kato, Nagatsu, Kimura & Sakakibara, 1978), alkaline phosphatase (Fernley & Walker, 1965), and a-glucosidase (Peters, Heath, Wansbrough-Jones & Doe, 1975) were assayed using published methods in brush-border and non-brush-border membrane fractions. Protein was assayed by the fluorescamine method (Udenfriend, Stein, Bohlen, Dairman, Leimgruber & Weigele, 1972).…”

Section: Cell Culturementioning

confidence: 99%

Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

Osypiw¹,

Gleeson²,

Lobley³

et al. 1994

Experimental Physiology

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SUMMARYAcid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16-to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes-or HCO3--buffered media. Intracellular pH (pH.) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mm K+, corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO3--free (Hepes-buffered) media, bilateral superfusion with 1 mm amiloride or with Na+-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4C1 pulse) by 86 and 98 % respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (I mM). The pH. recovery induced by apical Na+ readdition had a Michaelis constant (K,R) for Na+ of 30 mm and a relatively high inhibitor constant (K,) for amiloride of 45 5 /lM. Initial pHi in HCO3--buffered media was lower than in the absence of HCO3-(7 35 vs. 7 80). pH, recovery from an acid load in HCO3-was Na+ dependent but was inhibited only 18 % by I mM amiloride. The amiloride-independent pH. recovery was inhibited 49 % by pre-incubation of cells in 5 mm DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na+-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na+-H+ exchangers in several normal epithelia; and (b) a Na+-dependent HCO3-transport system, either Na+-HCO3-cotransport or Na+-dependent Cl--HCO3-exchange.

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“…Lazdunski et Ouellet ont etudik son action catalytique sur l'hydrolyse du phosphate de p-nitrophtnyle (I), sa sensibilite a certains inhibiteurs (2), et, en collaboration avec Brouillard, sa sensibi1itC aux variations de force ionique et de constante diklectrique (3). Fernley et Walker ont examin6 sa phosphorylation par quelques substrats (4), sa spkcificitk ( 5 ) , et sa catalyse de I'hydrolyse du phosnhate de mkthyl-4 umbellyphtryle (6). Quant B Fernley et Bisaz, ils ont ktudik sa phosphorylation par l'acide pyrophosphorique (7).…”

Section: Introductionunclassified

Etude cinétique des interactions électrostatiques entre la phosphatase alcaline intestinale de veau et ses substrats

Julien¹,

Ouellet²

1974

Can. J. Chem.

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Dc ;pnr.ter?~ent de Cl~ir?lie, Uni~vrsitP Lnvnl, Q~lPbec 10, Quebec GIK 7P4 R e~u le 15 fevrier 1974MARIUS JULIEX et L u~o v r c OUELLET. Can. J. Chem. 52, 3087 (1974). Le r6le de la charge de la niolCcule d'enzyme dans la cinetique de la reaction catalysee par diverses phosphatases alcalines a Cte etudie. Les donnees experimentales obtenues en utilisant le phosphate de p-nitrophenyle (charge nette -2 ) cornrne substrat montrent que la repulsion entre le molecule d'enzyme, de charge negative, et la molecule de substrat peut tenir compte, quantitativement, de la variation de la constante de Michaelis du systeme avec le p H entre p H 8.5 et p H 10.5. Toutefois les resultats obtenus en utilisant le phosphate d'ethanolamine (charge nette, -1) et dep-ou o-carboxyphenyle (charge nette -3) montrent soit que la partie organique de la molecule de substrat ne joue aucun r61e dans la formation du complexe enzyme-substrat, ou que la charge de la molecule d'enzyme ne joue pas le r6le suggere par les resultats obtenus avec le phosphate de p-nitrophenyle.Une etude plus pousske de cette derniere reaction en variant la force ionique et la constante dielectrique du milieu suggere que le centre actif de la molecule d'enzyme soit porteur d'une charge positive, et soit hydrophobe. MARIWS JULIEN and L U D~V I COUELLET. Can. J. Chem. 52, 3087 (1974). The role played by the ionic charge of the enzyme in the kinetics of the reactions catalyzed by alkaline phosphatases has been investigated. Data obtained from the hydrolysis ofp-nitrophenyl phosphatc (net charge -2 ) indicate that the repulsion between the negatively charged enzyme molecule and the substrate molecule is sufficient to take into account, quantitatively, the variation of the Michaelis constant with the p H o f the system between pH8.5 and 10.5. However, the results obtained with ethanolamine phosphate (net charge -I ) and p-or o-carboxyphenyl phosphate (net charge -3) show that either the organic part of the substrate molecule is not involved in the formation of the enzyme substrate intermediate or the charge of the enzyme does not have the role suggested from the work with p-nitrophenyl phosphate.Further investigation of this reaction in solutions of various ionic strength and dielectric constants suggests that the active center of the enzyme m~olecule is positively charged, but that hydrophobic effects are important. IntroductionLa phosphatase alcaline intestinale de veau (EC 3.1.3.1.)

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Kinetic behaviour of calf-intestinal alkaline phosphatase with 4-methylumbelliferyl phosphate

Cited by 180 publications

References 15 publications

Buffer-Induced Activation of Calf Intestinal Alkaline Phosphate

Buffer-Induced Activation of Calf Intestinal Alkaline Phosphate

Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

Etude cinétique des interactions électrostatiques entre la phosphatase alcaline intestinale de veau et ses substrats

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