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Purification and properties of molecular-weight variants of human placental alkaline phosphatase.

ABSTRACT: 1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8.6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4.2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.

SUBMITTER: Ghosh NK

PROVIDER: S-EPMC1198885 | biostudies-other | 1968 Aug

REPOSITORIES: biostudies-other

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Project description:Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

Project description:Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.

Project description:Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2, 4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were observed in reverse micelles. However, only one pKa value of 9.78-10.27 in aqueous solution was detected. Profiles of log V/K versus pL were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; at pL 10.4, the log V/K value was identical in water and deuterium. The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (pi) for deuterium for the transition state (piT) increased when the pH of the solution was raised. At pL 11.0, the piT was 1.07 in reverse micelles, which corresponds to the inverse-isotope effect of the reaction in this solvent system. Normal viscosity effects on kcat and kcat/Km were observed in aqueous solution, corresponding to a diffusional controlled physical step as the rate-limiting step. We propose that the rate-limiting step of the hydrolytic reaction changes from phosphate releasing in aqueous solution to a covalent phosphorylation or dephosphorylation step in reverse micelles.

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Purification and properties of molecular-weight variants of human placental alkaline phosphatase.

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