okadaic-acid and zinc-chloride

Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.

Topics: Acid Phosphatase; Aluminum; Amoeba; Animals; Calcium Chloride; Chlorides; Edetic Acid; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Magnesium Chloride; Okadaic Acid; Phosphoric Monoester Hydrolases; Tartrates; Zinc Compounds

Some lines of colon cancer cells are forced to undergo differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA). The increases in activities of both protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK) have been reported to be associated with the TPA-induced differentiation of HL-60 leukemia cells. In the present study, a 2-fold increase in PTP activity was observed in SW620 human colon cancer cells after 30 min of TPA treatment; a maximal level (4- to 5-fold) was reached at 60 min and continued for more than 6 hr. In addition, two TPA-induced differentiated characteristics, morphological alteration and release of cellular surface proteoglycan, were effectively blocked by PTP inhibitors, such as sodium orthovanadate (50 microM), zinc chloride (100 microM), and iodoacetate (250 microM), but not by the protein serine/threonine phosphatase inhibitor okadaic acid (20 nM). On the other hand, although TPA induced a transient slight increase in PTK activity (1.4-fold) at 60 min, four PTK inhibitors (genistein, herbimycin A, tyrphostin-23 and quercetin) had different effects on the TPA-induced release of cell surface proteoglycan. Genistein (60 microM) potentiated this process, but in contrast, quercetin (45 microM) could partially inhibit the TPA effect. Taken together, these observations suggest that both PTP and PTK activities were increased in SW620 cells in response to TPA; however, the activation of PTP seems to be preferentially required for the TPA-induced differentiation of SW620 human colon cancer cells.

Topics: Cell Differentiation; Chlorides; Colonic Neoplasms; Enzyme Activation; Ethers, Cyclic; Genistein; Humans; Isoflavones; Okadaic Acid; Protein Tyrosine Phosphatases; Proteoglycans; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Zinc Compounds

A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.

Topics: Calcium Chloride; Cell Nucleus; Chelating Agents; Chlorides; Citric Acid; Edetic Acid; Egtazic Acid; Enzyme Inhibitors; Heparin; Magnesium Chloride; Manganese Compounds; Molybdenum; Okadaic Acid; Phosphorylation; Pisum sativum; Protein Tyrosine Phosphatases; Sodium Fluoride; Spermine; Tartrates; Vanadates; Zinc Compounds

The src-related protein tyrosine kinase p56lck is thought to be important in regulating maturation and functional responsiveness of T cells and thymocytes. In the present studies we report that expression of p56lck is suppressed during apoptosis. Using primary cultures of rat thymocytes, we found that agents that are effective in inducing apoptosis, including okadaic acid, dexamethasone, and antibodies to the CD3 receptor, also deplete cells of p56lck. This process is rapid, occurring within 24 h, and is not due to cytotoxicity. Inhibition of DNA fragmentation in apoptotic cells with the endonuclease inhibitor ZnCl2 failed to prevent depletion of p56lck, suggesting that it was not a consequence of the DNA degradation process. Using the thymic lymphoma cell line LSTRA, apoptosis was also associated with cellular depletion of p56lck. In contrast to thymocytes, this process required 48-72 h possibly because these cells overexpress p56lck. Although at this time we are uncertain as to the precise role of p56lck in the process of apoptosis, our results indicate that changes in the expression of this protein in thymocytes is an important marker of programmed cell death.

Topics: Animals; Apoptosis; Chlorides; Dexamethasone; Ethers, Cyclic; Female; In Vitro Techniques; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Okadaic Acid; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Thymus Gland; Zinc Compounds