Modified Phosphatases
a technology of phosphatases and phosphates, applied in the field of phosphatases, can solve problems such as confusion of identity
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example 4
Preparation of Mutants
[0145]The mutants as herein in described and more specific the mutants as described in Tables 4, 5 and 6 are / were prepared by using standard molecular biology techniques.
[0146]The amino acid positions as mentioned in Tables 4 to 6 correspond to the sequences as depicted in FIG. 1. Only the mutated positions are indicated, i.e. only the deviations from the wild type sequences are given. For example mutant 1 of Table 4 is, if compared to the given wild type sequence, unchanged at positions 87, 93 and 429, i.e. position 87 is a K, position 93 is a G and position 429 is an E.
[0147]The mutants will be prepared and checked by standard molecular biology techniques, such as PCR site-directed mutagenesis, restriction enzyme analysis, and sequence analysis.
Example 5
[0148]At t=0 different recombinant alkaline phosphatases containing 450±50 Units were diluted 4000× in diluent buffer (0.025M glycine / NaOH pH 9.6 / 1 mM MgCl2 / 1% mannitol / 0.05% BSA) with different concentrations...
example 1
Dephosphorylation of the Biologically Active Substrate ATP by Different Phosphatases
[0155]ATP at a final concentration of 20 μM was incubated with different concentrations of BIAP, sALPP, sALPI or the chimera catALPI / crownALPP. From Table 9 it is obvious that pNPP chemical activity is not 1:1 related to the activity towards a biological substrate, e.g. ATP. Whereas BIAP and sALPI show more than 50% dephosphorylation of ATP after 90 minutes at 37° C. at concentrations of 0.031 and 0.004 pNPP units, respectively, ALPP and catALPP / crownALPP can only dephosphorylate this amount at concentrations of 0.125 and 0.0625 pNPP units, respectively.
example 2
Liver-Slice Assay Using Different Isoforms of Alkaline Phosophatase
[0156]Upon stimulation with LPS (10 μg / ml), liver slices produce NOx. FIG. 6 shows that in the presence of different recombinant human alkaline phosphatases (sALPI, sALPP, GPI-anchored ALPI, catALPI / crownALPP) at different concentrations, the NOx production was significantly inhibited. In this experiment, bovine derived ALPI was used as a positive control and solvent as a negative control.
[0157]Without the addition of LPS, NOx production by the liver slices was less then 10 μM and was not significantly altered by the presence of the different phosphatases during incubation (data not shown). From this it is concluded that all tested recombinant isoforms of human alkaline phosphatases, regardless of the presence of a GPI anchor have activity towards a biological substrate that is involved in the LPS-induced NOx production.
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