Isolation, Purification And Some Properties Of Hepatic Alkaline Phosphatase From Monopterus Albus

Alkaline phosphatase (ALP EC3.1.3.1) is one kind of enzyme,which is not absolutely specific.It could hydrolyze lots of phosphoric acid ester chemical compound in alkaline environment while the activator Mg²⁺ and Mn²⁺ are needed.It is widespread in prokaryotes and eukaryotes such as bacterium, alga,higher plant,insect,aquatic animal,mammal,people,etc.The wide distribution of ALP in organism suggests its essential role in cell phosphate metabolism,which is related to the absorption of phosphate and the biomineralization process in orgamisms.It's also a high valuable tool enzyme which can be used in studying nucleric acid,analysising nucleoticle sequences and the isolation, recombination of genes,blotting and ELISA,etc.The merchant reagent of ALP is very expensive.Monopterus albus (Zuiew) is an important economic fish.Comparing with activity of ALP in many species, we have found that there is high activity in the viscera of Monpererus.Albus. By thoroughly investigating and comparing the enzyme activity and isozyme of ALP in seven kinds of tissues from Monospters Albus(Zuiew).We have isolated and purified hepatic ALP and do some research on its enzymological properties and function groups composition.Now report the results of study as follow:1.Comparison study on ALP in seven kinds of tissues from Monospters Albus(Zuiew)The results indicated ALP exists in heart,serum,liver,kidney,cholecyst,spleen and intestines of Monopterus albus (Zuiew),the activity of ALP in different tissues are different,the activity in liver andcholecyst is strong but weak in serum.The electrophoretic patterns of ALP isoenzyme in these tissues have been studied by means of PAGE.The result indicatesd that the ALP isoenzyme patterns of Monopterus albus (Zuiew) exhibited tissue-specificity.Liver,kidney,cholecyst and spleen have one isozyme but intestines have two isozyme.2.Isolation and Purification of hepatic ALP from Monospters Albus(Zuiew)It was prepared and purified by means of the following techniques:n-butanol extraction,amona nium sulfate precipitation,ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Sephacryl S-200.Then the ALP obtained by Sephacryl S-200 gel chromatography was dialysed, dehydrated and identify the purification.The pure ALP showed one protein strip by PAGE,SDS-PAGE and IEF-PAGE,which indicate that pure ALP was homogen eous.Finally 21.93% of the ALP activity was recovered,ALP was 141.32-fold purified.The specific activity was 3021.39unit/mg protein.3. Characterization of hepatic ALP from Monospters Albus(Zuiew)The molecular weight of the enzyme is 133.4 kD and submit is 66.BkD as assayed by gel filtration chromatography on Sephacryl S-200 and SDS-PAGE.Isoelectric point of the pure ALP was 5.21 by IEF-PAGE.The kinetic characters of the enzyme have been studied.The optimum pH and optimum temperature for the enzyme to catalyze the hydrolysis of phenylphosphoric acid disodium salt are pH10.2 and 40°C,the Michaelis constant(ATw) is 1.17mmol/L and the maximum velocity(Vm) is 13.75umol/(L-min),the initial velocity(V0) is 6.02umol/(L-min).The activation energy of the enzyme is 52.6KJ-mol.The product HPO42' and the product-analog WO42" could competitively inhibit the activity of the enzyme.The positive monovalent alkali ions(k+,Na+)have no effects on the activity of the enzyme; Mg2+,Ca2+ activates the enzyme activity and Cu2+ ,Zn2+ inhibit the enzyme activity.The effectors methanol,ethanol,ethylene glycol and I-propyl alcohol inhibit the enzyme activity in varying degrees.The power of its inhibition is successivelyri-propyl alcohol >ethanol >methyl alcohol > ethylene glycoj.4.Function groups of hepatic ALP from Monospters Albus(Zu'K\v)The alkaline phosphatase were selectively modified by PMSF,DTT,PCMB,NBS,TNBS,SUAN...