Studies on Hydrolysis of P-Nitrophenyl Picolinate by Unsymmetrical Bis-Schiff Base Manganese Complexes with Aza-Crown Ether or Morpholino Pendants

The unsymmetrical bis-Schiff base manganese complexes with either benzo-10-aza-crown ether pendants ( MnL1Cl, MnL2Cl) or morpholino pendant (MnL3Cl,MnL4Cl) have been employed as models for hydrolase by studying the hydrolysis reactions of p-nitrophenyl picolinate (PNPP). The effects of complex structures, reaction pH and reaction temperature on the rate of PNPP hydrolysis have been investigted. All four complexes exhibit high catalytic activity and the rate increases with pH increasing at 25 oC. The complexes of ligands containing a crown ether group exhibit higher catalytic activities than the non-crown analogues.

Comparative Study of p-nitrophenyl picolinate Hydrolysis Catalyzed by Bis(O,O’-di(2-phenylalkyl)dithiophosphate) nickel(II)

The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by two nickel (II) complexes (bis(O,O’-di(2-phenylmethyl) dithiophosphate) nickel(II) (NiR1) and bis(O,O’-di(2-phenylethyl) dithiophosphate) nickel(II) (NiR2)) was investigated kinetically in this work. The results indicate that both metal complexes accelerate the hydrolysis of PNPP dramatically and the NiR1 exhibits higher catalytic function on PNPP hydrolysis in the buffered solution with relatively low pH values, while NiR2 shows slightly more efficacy on hydrolysis of PNPP in relatively high pH buffered solutions. This variance is ascribed to the synergism effect of space hindrance of the complexes and the nucleophilic attack of metal-hydroxy species generated by the complexes.

Water stress inhibits p-nitrophenyl phosphate hydrolysis activity of the plasma membrane H+-ATPase from soybean hypocotyls

The influence of water stress on ATP and p-nitrophenyl phosphate (PNPP) hydrolysis by plasma mem-brane ATPases was investigated using plasma membrane vesicles purified from soybean hypocotyls by the sucrose gradient centrifugation method. Results showed that ATPase activity was reduced after 10% polyethylene glycol (PEG) 6000 treatment for 12 h. Water stress also moved the optimal pH from 6.5 to 7.0. A significant decrease in PNPP hydrolysis was observed under PEG treatment. The Km for PNPP hydrolysis was shifted from 2.3042 0.0009 to 2.5048 0.0346 mmol L –1 . Moreover, PNPP hydrolysis was more sensitive to vanadate after PEG treatment, while inhibition of ATP hydrolysis by hydroxylamine was not affected. Our experimental results indicated that water stress changed the catalytic mechanism of the plasma membrane H + -ATPase through affecting the dephosphorylation process catalysed by its phosphatase domain.

Ecto-Phosphatase Activities on the Cell Surface of the Amastigote Forms of Trypanosoma cruzi

Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phos-pho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hy­drolysed at a rate of 80 nmol ·mg -1 ·h -1 in the presence of 5 mм MgCl2, pH 7.2 at 30 °C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mм MgCl2· Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mм ) . In the absence of Mg2+ (basal activity) the stimulating half concentration (S0. 5) for PNPP was 1.57 mм , while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg2+-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mм . The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phos-phothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-aminoacids and phosphoproteins under physiological conditions.

Role of cyclic GMP in cholinergic activation of Na-K pump in duck salt gland

Na-K-ATPase will hydrolyze an alternate substrate, p-nitrophenylphosphate (pNPP). The hydrolysis is ouabain sensitive and occurs at an external site on the cell membrane, thereby allowing measurement of Na-K-ATPase activity in the intact cell. pNPP hydrolysis was monitored in salt gland slices incubated in a bicarbonate-buffered Ringer solution. Methacholine-stimulated pNPP hydrolysis was inhibited by either atropine or ouabain. The hydrolysis was also dependent on the presence of calcium and sodium in the Ringer solution. cGMP stimulated ouabain-sensitive pNPP hydrolysis at concentrations from 10(-8) M to 10(-4) M with an optimum at 10(-5) M. cAMP did not produce a significant activation of pNPP hydrolysis. The effect of cGMP ws not dependent on either sodium or calcium in the Ringer solution and was not inhibited by atropine. Hydrolysis of pNPP promoted by either methacholine or cGMP occurred under conditions where no net influx of Na+ into the cells could occur. Sodium-pump activation during cholinergic stimulation, therefore, does not depend on an elevation of cell sodium but must occur by another mechanism. cGMP appears to play a direct role in that mechanism.