Phosphoester specificity of purified human liver alkaline phosphatase

1979 ◽  
Vol 57 (7) ◽  
pp. 1000-1007 ◽  
Author(s):  
L. E. Seargeant ◽  
R. A. Stinson

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase.The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.

Author(s):  
Л.А. МАРЧЕНКО ◽  
Т.Н. БОКОВИКОВА ◽  
Е.В. ЛИСОВАЯ ◽  
С.А. ИЛЬИНОВА ◽  
Е.П. ВИКТОРОВА

Исследована возможность применения яблочной кислоты (ЯК) в качестве гидратирующего агента для перевода негидратируемых форм фосфолипидов в гидратируемые. Выбор ЯК обусловлен ее стоимостью, которая в 1,5 раза меньше стоимости янтарной кислоты, широко применяемой в качестве гидратирующего агента, а также большей доступностью с точки зрения промышленного производства. Исследование процесса комплексообразования и определение состава комплексных соединений ЯК с ионами кальция и магния осуществляли методом потенциометрического титрования. Установлено, что внесение в раствор ЯК ионов кальция и магния приводит к снижению значений рН, что свидетельствует о наличии комплексообразования в указанных системах. Наиболее устойчивыми являются комплексы ионов кальция и магния с непротонированным лигандом при соотношении Ме2 : лиганд 1 : 1. В процессе комплексообразования ионы Ca2 и Mg2 вытесняют протоны только карбоксильных групп ЯК, которая с ионами кальция образует более устойчивые комплексы, чем с ионами магния. Показано, что устойчивость комплексов ЯК с ионами кальция и магния значительно выше, чем устойчивость комплексов фосфатидилсеринов и фосфатидных кислот с указанными ионами. Использование водных растворов ЯК в качестве гидратирующего агента позволит повысить эффективность процесса гидратации и увеличить выход фосфолипидов и, следовательно, готового продукта лецитина. The possibility of using malic acid (MA) as a hydrating agent for converting non-hydrated forms of phospholipids into hydrated ones has been investigated. The choice of MA is due to its cost, which is 1,5 times less than the cost of succinic acid, as well as greater availability from the point of view of industrial production. The study of the complexation process and determination of the composition of complex compounds of MA with calcium and magnesium ions was carried out by the method of potentiometric titration. It was found that the introduction of calcium and magnesium ions into the MA solution leads to a decrease in pH values, which indicates the presence of complexation in these systems. Complexes of calcium and magnesium ions with an unprotected ligand at a ratio of ME2 : ligand 1 : 1 are the most stable. In the process of complexing, Ca2 and Mg2 ions displace protons only of the carboxyl groups of MA, which forms more stable complexes with calcium ions than with magnesium ions. It is shown that the stability of MA complexes with calcium and magnesium ions is significantly higher than the stability of phosphatidylserine and phosphatidic acid complexes with these ions. Using water solutions of MA as a hydrating agent will increase the efficiency of the hydration process and increase the yield of phospholipids and, consequently, the finished product lecithin.


2006 ◽  
Vol 19 (3) ◽  
pp. 683-696 ◽  
Author(s):  
N. Choucair ◽  
V. Laporte ◽  
R. Levy ◽  
C. Tranchant ◽  
J.-P. Gies ◽  
...  

1960 ◽  
Vol 43 (6) ◽  
pp. 1149-1169 ◽  
Author(s):  
M. Kunitz

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.


1972 ◽  
Vol 127 (1) ◽  
pp. 87-96 ◽  
Author(s):  
P. G. Bolton ◽  
A. C. R. Dean

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.


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