Improved method for estimation of inorganic phosphate: implications for its application in enzyme assays.

The conventional method of Fiske and Subba Row for the estimation of inorganic phosphate (Pi) is although rapid, but suffers from the disadvantage that the color is unstable and hence the optical density (OD) measurements have to be carried out within a short time span of 8-12 min. This poses a restriction on the number of samples, which can be handled in a batch. Although, modified procedures involving use of alternate reducing agents/or increasing the concentration of H2SO4 in conventional method have been subsequently developed, but the problem of color stability could not be solved. In addition, the use of higher concentrations H2SO4 has rendered the methods unsuitable in enzyme assays, especially if the acid labile phosphate containing substrates have been used. In the present study, attempts have been made to suitably modify the method to improve the stability of the color and sensitivity and also for its applicability in enzyme assays, especially when acid labile phosphate containing substrates such as ATP is used. We used the higher concentrations (0.625, 0.8 and 1.0 N) of H2SO4 rather than 0.5 N used in the conventional assay procedures. Under these conditions, the reagent blanks do not develop color for up to 24 h, whereas the intensity of the molybdenum blue color in the standard and/or experimental tubes increased with time reaching optimum value at 24 h. Simultaneously, the absorption maximum shifts from 660 nm to 820 nm. The highest concentration of H2SO4 (1.0 N) is found to be the most effective in the process of color development. The sensitivity of the method is from 1.7 to 2.1 times higher, as compared to the conventional Fiske and Subba Row method for the measurements carried out at the end of 15 min at 820 nm and with the highest concentration of H2SO4 (1.0 N); the sensitivity increased 4.8-fold at the end of 24 h. Presence of glucose and sucrose (1-10 mM), NaCl and KCI (5-100 mM), MgCl2 (1-10 mM) and BSA (10 to 500 microg per assay tube) do not interfere either with color development or with OD measurements. The extent of ATP hydrolysis is 1.6 to 3.4% for up to 1 hi, depending upon the concentration of H2SO4 used. Only negligible hydrolysis of G6P is observed under these conditions. These results suggest that the presently modified method is suitable for Pi analysis in the enzyme assays, in the presence of labile phosphate containing substrates.

Effect of repeated intraperitoneal exposure to picrotoxin on rat liver lysosomal function.

Effect of repeated (20 days) exposure to picrotoxin (PTX) on rat liver lysosomal function was evaluated by measuring the free and total activities of acid phosphatase, cathepsin D, ribonuclease II (RNAse II) and deoxyribonuclease II (DNAse II). The free activities of the nucleases (both RNAse II and DNAse II) were increased following PTX exposure. The total DNAse II activity was increased by 2.2-fold whereas the total acid phosphatase activity was decreased by 28%. Consequently, the ratios of total activity / free activity were low in the PTX exposed groups, implying loss of membrane integrity. Cathepsin D activity was completely abolished. The results show that repeated exposure to PTX can lead to lysosomal dysfunction in liver.

Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates.

Kinetic properties of rat liver acid phosphatase were evaluated using the conventional synthetic substrates sodium beta glycerophosphate (betaGP) and p-nitrophenyl phosphate (PNPP) and physiologically occurring phosphate esters of carbohydrates, vitamins and nucleotides. The extent of hydrolysis varied depending on the substrates; phosphate esters of vitamins and carbohydrates were in general poor substrates. Kinetic analysis revealed the presence of two components of the enzyme for all the substrates. Component I had low Km and low Vmas. Opposite was true for component II. The Km values were generally high for betaGP, PNPP and adenosine diphosphate (ADP). Amongst the nucleotides substrates AMP showed high affinity i.e. low Km. The increase in enzyme activity in general at high substrate concentration seems to be due to substrate binding and positive cooperativity. AMP which showed highest affinity was inhibitory at high concentration beyond 1 mM. The results suggest that in situ the nucleotides may be the preferred substrates for acid phosphatase.