Alkaline phosphatase from pig kidney. Method of purification and molecular properties.
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ABSTRACT:
SUBMITTER: Wachsmuth ED
PROVIDER: S-EPMC1168074 | biostudies-other | 1974 Jul
REPOSITORIES: biostudies-other
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Project description:The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (V(max.)) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme-substrate complex is 8.7 for p-nitrophenyl phosphate and beta-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme-substrate complex, 8.7, is very similar to that for the enzyme-substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (DeltaH) at pH9.5 showed a transition at 24.8 degrees C that was unaffected by Mg(2+). 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn(2+) ions/tetrameric enzyme for an ordered association of the monomers. Zn(2+) in excess and other bivalent ions compete for a second site with Mg(2+). Mg(2+) enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.
| S-EPMC1168075 | biostudies-other
Project description:Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.
| S-EPMC1172889 | biostudies-other
Project description: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.
| S-EPMC1198885 | biostudies-other
Project description:1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.
| S-EPMC1177276 | biostudies-other
Project description:1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0 degrees . 2. Inactivated enzyme can be partially reactivated by incubation at 30 degrees in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3.3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.
| S-EPMC1198799 | biostudies-other
Project description:The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.
| S-EPMC1161134 | biostudies-other
Project description:Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg(2+) and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of K(m) and V(max.) were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.
| S-EPMC1168076 | biostudies-other
Project description:The mechanisms by which phosphate regulates the activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) in rat kidney were investigated. Measurements of incorporation of [(14)C]leucine into kidney alkaline phosphatase in rats fed on complete or phosphate-free diet provide evidence of a twofold increase in the rate of synthesis of the enzyme in diet-treated animals. Cycloheximide experiments indicated that control and diet-adapted enzyme decreases in activity according to first-order kinetics with a calculated half-life of 10.3 and 6.5h after complete and phosphate-free diet administration respectively. Basal and diet-adapted enzymes exhibit similar K(m) values for several phosphomonoesters and an identical degree of inhibition is produced by cysteine. In addition, the enzyme from both sources is the same with regard to heat inactivation at 45, 56 or 64 degrees C, to the profile of elution from Sephadex and to electrophoretic properties on polyacrylamide gel. A failure of rat kidney alkaline phosphatase to respond to cortisol (hydrocortisone) was also observed.
| S-EPMC1176483 | biostudies-other
Project description:The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg(2+) ions was investigated with the aid of kinetic measurements. Mg(2+) ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg(2+) ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg(2+) must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg(2+) ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg(2+), it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.
| S-EPMC1168072 | biostudies-other
Project description:PURPOSE OF REVIEW:In chronic kidney disease (CKD), disturbance of several metabolic regulatory mechanisms cause premature ageing, accelerated cardiovascular disease (CVD), and mortality. Single-target interventions have repeatedly failed to improve the prognosis for CKD patients. Epigenetic interventions have the potential to modulate several pathogenetic processes simultaneously. Alkaline phosphatase (ALP) is a robust predictor of CVD and all-cause mortality and implicated in pathogenic processes associated with CVD in CKD. RECENT FINDINGS:In experimental studies, epigenetic modulation of ALP by microRNAs or bromodomain and extraterminal (BET) protein inhibition has shown promising results for the treatment of CVD and other chronic metabolic diseases. The BET inhibitor apabetalone is currently being evaluated for cardiovascular risk reduction in a phase III clinical study in high-risk CVD patients, including patients with CKD (ClinicalTrials.gov Identifier: NCT02586155). Phase II studies demonstrate an ALP-lowering potential of apabetalone, which was associated with improved cardiovascular and renal outcomes. SUMMARY:ALP is a predictor of CVD and mortality in CKD. Epigenetic modulation of ALP has the potential to affect several pathogenetic processes in CKD and thereby improve cardiovascular outcome.
| S-EPMC6903384 | biostudies-literature