Analysis phosphatase inhibition

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

As for the methods of analysis, the regulation considers biologic methods (bioassay in mice and in rats) and alternative methods [high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and phosphatase inhibition]. When the results of the analyses performed demonstrates discrepancies between the different methods, the mouse bioassay should be considered as the reference method. 

Another variant of PP2A assay is the one reported by Isobe et al. [166] where a firefly bioluminescence system is used for the detection of protein phosphatase 2A inhibitors, in which luciferin phosphate is hydrolyzed to luciferin and inorganic phosphate by protein phosphatase 2A. The recent commercial availability of the phosphatase enzymes, which obviates the need to isolate them from animal tissues, also makes this approach very attractive. However, not all microcystins variants react with protein phosphatase enzymes to a similar extent [161,163] and the assay is sensitive to protein phosphatase inhibitors other than microcystins, such as okadaic acid, tautomycin, and calyculin A. In addition, the cyanobacterial sample itself may contain phosphatase activity that masks the presence of toxins [160]. As a consequence, the lack of specificity of the protein phosphatase inhibition assays requires that additional confirmatory analytical methods be employed for specific analysis of cyanobacterial toxins. 

Osterhages work has led to further analysis of bioactive products from the marine fungus A. salicorniae. In addition to the novel tetramic acid derivatives (38, 39), this fungus also produces two new epimeric lactones, the cycloethers ascolactone A (43) and B (44), similar to known cephalosporide compounds, and ascochitine (45), ascochital (46), and halopyrone (47), which were previously known.Compound 45 exhibited moderate inhibition of a tyrosine phosphatase B from Mycobacterium tuberculosis. 

AGIRE computer program for, 249, 79-81, 225-226 comparison to analysis based on rates, 249, 61-63 complex reactions, 249, 75-78 experimental design, 249, 84-85 inhibitor effects, 249, 71-75 potato acid phosphatase product inhibition, 249, 73-74 preliminary fitting, 249, 82-84 prephenate dehydratase product inhibition, 249, 72-73 product inhibition effects, 249, 72-73 prostate acid phosphatase phenyl phosphate hydrolysis, 249, 70 reactions with two substrates, 249, 75-77 reversible reactions, 249, 77-78 with simple Michaelian enzyme, 249, 63-71 [fitting equations, 249, 63] with slow-binding inhibitors, 249, 88 with unstable enzymes, for kinetic characterization, 249, 85-89. 

Prostatic acid phosphatase is partially and reversibly inactivated by calcium ion (45). Anions such as chloride, bromide, and thiocyanate inhibit prostatic acid phosphatase competitively with regard to substrate as well as noncompetitively. A kinetic analysis by London et al. (46) indicates that the noncompetitive inhibition was related to changes in charge on the protein molecule. A variety of nonspecific anions accelerate thermal denaturation of the enzyme. The enzyme is quite sensitive to a number of electrolyte changes, but it is not clear whether these factors are involved in biological control. 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

Thompson KM, Uetani N, Manitt C et al (2003) Receptor protein tyrosine phosphatase sigma inhibits axonal regeneration and the rate of axon extension. Mol Cell Neurosci 23 681-92 Tobaben S, Sfldhof TC, Stahl B (2002) Genetic analysis of a-latrotoxin receptors reveals functional interdependence of CIRL/latrophilin 1 and neurexin la. J Biol Chem 277 6359-65 Tobaben S, Sfldhof TC, Stahl B (2000) The G protein-coupled receptor CL1 interacts directly with proteins of the Shank family. J Biol Chem 275 36204-10 Tonks NK (2006) Protein tyrosine phosphatases from genes, to function, to disease. Nat Rev Mol Cell Biol 7 833 16... 

Both mPA-PLAjU and mPA-PLAj/ , like many phosphate-recognizing enzymes such as phosphatase and ATPase [40], are inhibited by vanadate [51]. When expressed in insect Sf9 cells, neither mPA-PLAja nor mPA-PLAj/ were secreted. Both were recovered from the Triton X-lOO-insoluble fraction of the cell membrane. An immunofluorescence analysis confirmed that these proteins are localized exclusively to the plasma membrane. Both mPA-PLAja and -fi may locahze in the detergent-resistant membrane domain, referred to as raft structures, and function as lysoPA-producers under certain conditions [51]. 

Alkaline phosphatase exists in two forms in animal tissues, one activated by added Mg(II) or Mn(II) and not inhibited by F or CN ions, the other independent of added divalent ions but inhibitable by cyanide . Yeast phosphatase, active at neutral pH, is activated by divalent metal ions in the order Mg > Mn > Ni, Co, Fe ". Metal analysis of purified kidney phosphatase reveals a mixture of bound ions Zn, Cu, Mn, Mg, and Fe . This variety of responses illustrates a classical problem in studies of enzyme activation by metal ions defining the species that is of greatest importance in vivo. 

The total number of polypeptides in the culture medium was 8-20 bands according to electrophoretic analysis (Figure 1). The protein export was inhibited by actinomycin D, cycloheximide, 2-deoxy-D-glucose and also by concanavalin A (Con A). In the presence of Con A (400 pg/ml), the enzymes were accumulated entirely inside the cells but not in the periplasm or the cell wall (Figure 2). At the same time the lectin had no influence upon cell growth and expression of invertase and acid phosphatase associated with the cell envelope. The activity accumulated inside Con A - treated cells was released into the culture medium on a-methylmannoside addition (Figure 3). The process was not affected by cycloheximide but was inhibited by sodium azide. The effect of Con A appears to be conditioned by its interaction with the plasma membrane of intact cells. Besides Con A, we detected peroxidase interaction with the plasmalemma of intact cells and alkaline phosphatase internment under physiological conditions. 

---