Svetlana M. Avaeva

A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay.

An improved procedure for phosphate determination based on a highly colored complex of phosphomolybdate and malachite green is described. All necessary reagents are combined in one concentrated solution, making the assay sensitive and convenient. The procedure is based on the finding that the dye is easily soluble and stable in the presence of 6 N acid. The addition of Tween 20 is required to stabilize the dye-phosphomolybdate complex at phosphate concentrations above 10 microM. The time of color development at 25 degrees C is about 3 min. The procedure was adopted to measure alkaline phosphate activity in heterogeneous enzyme immunoassay with rho-nitrophenyl phosphate and pyrophosphate as substrates. In both cases, a 4-fold increase in sensitivity in terms of absorbance readings was obtained compared to the standard method based on rho-nitrophenol measurement. In visual analysis, the gain in sensitivity was as high as 20-fold, due to contrast color change (yellow to greenish blue).

A simple and sensitive apparatus for continuous monitoring of orthophosphate in the presence of acid-labile compounds

Abstract An inexpensive analyzer for continuous recording of orthophosphate concentration has been designed utilizing instruments used in routine liquid chromatography. The determination is based on the change in the spectrum of methyl green upon binding to molybdophosphoric acid (H. Van Belle, 1970 , Anal. Biochem. 33 , 132–142). Changes in P 1 concentrations down to 1 and 5 μ m in a sample can be displayed full-scale on the recorder at sample consumption rates of 7 and 1.4 ml/min, respectively. Short time of sample contact with acid (down to 6 s) enables measurements to be made in the presence of highly labile compounds such as acetyl phosphate.

X‐Ray crystallographic studies of recombinant inorganic pyrophosphatase from Escherichia coli

An E. coli inorganic pyrophosphatase overproducer and a method for a large‐scale production of the homogeneous enzyme are described. The inorganic pyrophosphatase was crystallized in the form containing one subunit of a homohexameric molecule per asymmetric unit: space group R32, a = 110.4 Å, c = 76.8 Å. The electron density map to 2.5 Å resolution phased with Eu‐ and Hg‐derivatives (figure of merit, = 0.51) was improved by the solvent flattening procedure ( = 0.77). The course of the polypeptide chain and the secondary structure elements, intersubunit contacts and positions of the active sites were characterized. Homology with S. cerevisiae inorganic pyrophosphatase structure was found.

Inorganic pyrophosphatase as a label in heterogeneous enzyme immunoassay.

Inorganic pyrophosphatase from Escherichia coli has been employed as a label in heterogeneous enzyme immunoassays. Enzyme-antibody conjugates were prepared with the use of glutaraldehyde and purified by gel permeation chromatography. Enzyme activity was measured by means of a sensitive one-step color reaction between phosphate, molybdate, and malachite green. The sensitivity in terms of absorbance readings was four to eight times higher than that of peroxidase-based assays. The color change (yellow to greenish blue) inherent in the use of pyrophosphatase as the labeling agent is highly suitable for visual analysis. Other merits of pyrophosphatase include the remarkable stability of the enzyme and its substrate, its compatibility with bacteriostatic agents, and its low Michaelis constant. Examples of the use of phosphatase in the assay of human alpha-fetoprotein and immunoglobulin G are presented.

A comparative study of carbohydrase activities in marine invertebrates

Abstract 103 species of marine invertebrates, belonging to 7 types Spongia, Coelenterata, Annelida, Arthropoda, Mollusca, Echinodermata, Chordata, have been tested for β-1,3-glucanase (laminarinase), β-1,4-glucanase (cellulase) and amylase activities.

Isolation, subunit structure and localization of inorganic pyrophosphatase of heart and liver mitochondria

A procedure has been developed to isolate separately two forms (I and II) of inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from bovine heart mitochondria with specific activities of 250 and 39 IU/mg, respectively. The values of Mr for enzymes I and II are about 60000 and 185000, respectively. Polyacrylamide gel electrophoresis of pyrophosphatase II in the presence of sodium dodecyl sulfate reveals polypeptides of four types with Mr of 28000 (alpha), 30000 (beta), 40000 (gamma) and 60000 (delta). Enzyme I consists of two subunits similar in mass to alpha and beta. When rat heart and liver mitochondria are fractionated with digitonin and Lubrol WX, pyrophosphatase II, but not I, remains bound to inner membrane fragments. The results show that the two forms of the mitochondrial pyrophosphatase, one of which is localized in the inner membrane, differ in subunit structure but have a common catalytic part.

Fluoride inhibition of inorganic pyrophosphatase. IV. Evidence for metal participation in the active center and a four-site model of metal effect on catalysis.

Atomic spectroscopy of native yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) after gel filtration showed that it only binds activating Mg2% in an easily dissociable manner. Formation of a covalent intermediate between the enzyme and an entire substrate molecular in the presence of fluoride, however, dramatically strengthened the binding of two Mg2+ per subunit and eliminated at neutral pH the effect of added metals on protein fluorescence but not on the absorption spectrum, suggesting that different mental binding sites influence the two spectra. This conclusion was confirmed by spectra studied on native enzyme. A third, low-affinity site for Mg2+ was found on the enzyme pH greater than 8. A model of enzyme-substrate-metal interactions was proposed, according to which the fluorescence-controlling site belongs to the active center and substrate can only be bound to it as a 1 : 1 complex with metals.

Studies on inorganic pyrophosphatase using imidodiphosphate as a substrate

Bakers yeast inorganic pyrophosphatase has been found to catalyze Mg2+‐dependent hydrolysis of imidodiphosphate yielding phosphate and amidophosphate. The reaction proceeds linearly in the presteady state. The catalytic constant is maximal at pH 9.0 and equals 0.5 min−1. Kinetic titrations of the enzyme with imidodiphosphate and Mg2+ have provided direct evidence for the involvement of three Mg2+ per active site in the transition state of the pyrophosphatase reaction.

Escherichia coli inorganic pyrophosphatase : site-directed mutagenesis of the metal binding sites

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the κ app value by approx. 2–3 orders of magnitude. At the same time, the K m values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild‐type pyrophosphatase.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4(2-). Ligand-induced molecular asymmetry.

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.