Isolation, Purification And Some Properties Of Intestinal Alkaline Phosphatase From Hypophthalmichthys Molitrix

Alkaline phophatase (ALP EC3.1.3.1) exists in various organisms from bacteria to mammals. 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 organisms. It's also a high valuable tool enzyme which can be used in studying nucleic acid, analysising nucleic sequences and the isolation, recombination of genes, blotting and ELISA, et al. On the other hand, it has other uses such as those in biosensors, cosmetic and Therapeutic Drug. In clinic, serum ALP is the warrant that is used to diagnose some disease.Presently many scholars have studied ALPs in various seashells and fishes, but it haven't been reported that the ALP in non-stomach fish. Hypophthalmichthys molitrix is an important economic fish. In our laboratory, comparing with activity of ALP in many tissues, we found that there is high activity in the intestines of Hypophthalmichthys molitrix .By investigating and comparing the enzyme activity in seven kinds of tissues from. We isolated and purified intestinal ALP and do some research on enzymological properties and function groups.Now report the results of study as flow:1. Comparison study on ALP in seven kinds of tissues from Hypophthalmichthys molitrix (silver carp)The results indicated ALP existing in heart, intestines, kidney, liver of Hypophthalmichthys molitrix, the activity of ALP in different tissue is different, and the activity in intestines is strong.2. Isolation and Purification of intestinal ALP from Hypophthalmichthys molitrix (silver carp)It was prepared and purified by means of the flowing techniques: n-butanol extraction, amonanium sulfate precipitation, Ultra filtration, ion exchange chromatography on DEAE-sepharose Fast Flow column and gel filtration chromatography on Superdex-200. Then the pure ALP obtained by Superdex-200 gel chromatography was dialyzed, dehydrated and identify the purification. The ALP was 105.08-fold purified. The specific activity was 4031.15unit/mg protein. 3. Characterization of intestinal ALP from Hypophthalmichthys molitrix (silver carp)The molecular weight of the enzyme is 135.4kD and submit are 67.3kDa as assayed by gel filtration chromatography on Superdex-200 and SDS-PAGE. Isoelectric point of pure ALP was 5.31 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 pH 10.2 and 25℃, the Michaelis constant(Km) is 0.473mmoJ/L and the maximum velocity (Vm)is 14.35μmol/ (L·min).The product HPO^2- and the product-analog WO^2- could competitively inhibit the activity of the enzyme, the positive monovalent alkali ions(K⁺, Na⁺) have no 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 successively: I-propyl alcohol> ethanol> methanol> ethylene glycol4. Function groups of intestinal ALP from Hypophthalmichthys molitrix (silver carp)The alkaline phosphatase were selectively modified by PCMB,NBS,PMSF,SUAN,DTT and IAc after that the changes in their activities have been detected, It was found that the reaction of ALP with PMSF, NBS,TNBS,SUAN,DTT and IAc resulted in a strong inhibition of enzyme activities which decreased with the increase if modifier concentration. The activity of ALP with p-chloromercuribenzoic acid has been no inhibition. The results show that Ser, Lys and Trp residues are essential functional groups of the ALP. Partial disulfide bond are essential, it also for the function of this enzyme.