Jukka Heinonen

A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase

Abstract A new and convenient method for the determination of P i was developed. Phosphomolybdate is measured colorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acid-labile phosphates (e.g., creatine phosphate) causes no problems, because extra molybdate is complexed with citrate immediately after the color has been developed. Strong reductants and SH compounds which interfere, if present in high concentrations, are eliminated by adding H 2 O 2 . Detergents, organic bases, protein, and sucrose do not interfere. The assay is as sensitive as most modifications of the Fiske-SubbaRow method. In the routine procedure the useful range is 50–1500 nmol of P i . The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.

Conservation of functional residues between yeast and E. coli inorganic pyrophosphatases

The alignments of the amino acid sequences of inorganic pyrophosphatase (PPase) from Saccharomyces cerevisiae (Y1-PPase, 286 amino acids) and Escherichia coli (E-PPase, 175 amino acids) are examined in the light of crystallographic and chemical modification results placing specific amino acid residues at the active site of the yeast enzyme. The major results are: (1) the full E-PPase sequence aligns within residues 28-225 of Y1-PPase, raising the possibility that the N-terminal and C-terminal portions of Y1-PPase may not be essential for activity, and (2) that whereas the overall identity between the two sequences is only modest (22-27% depending on the choice of alignment parameters), of some 17 putative active site residues, 14-16 are identical between Y-PPase and E-PPase. PPase thus appears to be an example of enzymes from widely divergent species that conserve common functional elements within the context of substantial overall sequence variation.

A method for the concentration and for the colorimetric determination of nanomoles of inorganic pyrophosphate.

Abstract A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate ( PP i ) was developed. PP i was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl 2 and KF. The separated PP i was dissolved by immersing the filter in 0.5 n H 2 SO 4 . Inorganic phosphate ( P i ) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PP i was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PP i can be accurately assayed even when there is a 10 4 -fold excess of P i . Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PP i . Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PP i in the same sample.

New radiochemical method for assay of enzymes catalyzing the cleavage of inorganic pyrophosphate

Abstract A new method has been developed for the assay of pyrophosphatases. Inorganic pyrophosphate labeled with 32P is used as substrate and after the reaction the remaining pyrophosphate is precipitated as the manganous salt at pH 4.0. Phosphate formed from the labeled pyrophosphate in the enzyme reaction remains in solution. Therefore the radioactivity of the solution after separation of the precipitate by centrifugation is linearly proportional to the activity of the pyrophosphatase in the reaction mixture. The main advantages of this radio-chemical method over the colorimetric one are: 1. (a) The degree of precipitation is independent of time, whereas in the development of the color of molybdenum blue time must be strictly controlled. 2. (b) The radiochemical method is insensitive to the presence of many compounds (labile phosphate esters, reducing compounds, etc.) which may disturb the assay of pyrophosphatases by the colorimetric method. 3. (c) It also measures the phosphorylation of sugars at the expense of inorganic pyrophosphate, a reaction catalyzed by some pyrophosphatases.

Intracellular concentration of inorganic pyrophosphate in the cells of Escherichia coli: A method for its determination

Abstract A new method was developed for the separation of labelled inorganic pyrophosphate (PP 1 ) from the extracts of bacterial cells grown in the presence of labelled phosphate of high specific activity. First PP 1 is precipitated as manganous salt. Further purification is achieved in two chromatographic runs performed in the opposite directions on the same sheet of paper. The first solvent system consists of ethyl methyl ketone-methanol-water-36% HCl (20:40:10:1, by vol) and the second of ethyl methyl ketone-methanol-water-trichloroacetic acid-25% NH 4 -EDTA (40 ml: 10 ml: 15 ml: 5 g: 0.8 ml: 50 mg). Each run takes only about 40 min of time. Thus it is possible to treat tens of samples in one working day. Using this procedure the intracellular concentration of PP 1 in the exponential phase cells of Escherichia coli growing aerobically in minimal medium at 37°C was determined to be about 1.3 m m or 5.5 nmoles/mg of dry weight.

Activity Changes of Inorganic Pyrophosphatase of Streptococcus faecalis during Batch Culture

The state of inorganic pyrophosphatase (EC 3.6.1.1) from Streptococcus faecalis ATCC 8043 was studied in different phases of batch culture. The degree of inactivation (i.e. the ratio of activities observed before and after incubation at 37 degrees C without cysteine) was highest, and the degree of activation (i.e. the ratio of activities after and before incubation in the presence of cysteine) was lowest, in samples taken during the early-exponential growth phase. During the various phases of batch culture, the specific activity before incubation and the degree of inactivation changed in parallel, whereas the specific activity observed after incubation remained nearly constant. During the early-exponential phase of growth almost all the enzyme was in the high-activity form, whereas during the stationary phase the highly active and the less active forms existed in equal amounts. These findings suggest that inorganic pyrophosphatase in S. faecalis is synthesized constitutively and is primarily regulated at the level of activity.

Biological Production of PPi

Inorganic pyrophosphate (PPi) was discovered already in the nineteenth century. As its name implies, it is synthesized by heating sodium or potassium salts of orthophosphate (see van Wazer 1958). Formation of PPi in a biological system was reported in 1941 by Cori, who found that it accumulated in rat liver extract incubated aerobically in the presence of succinate and fructose (referred in Cori et al. 1951). The first biological reaction, where PPi was formed was described by Kornberg in 1948. He found that yeast cell extract catalyzed formation of ATP and NMN in the presence of NAD+ and PPi. He proposed the name pyrophosphorolysis for this reaction in analogy with the already known phosphorolysis. The reaction was readily reversible and thus PPi and NAD+ were the products, if ATP and NMN were used as substrates. In the 1950s many similar reactions were observed, and in 1957 Kornberg proposed in a review article that in vivo pyrophosphorylases mostly act in the direction of PPi formation serving the biosynthesis of stable biochemical compounds. Coupling to hydrolysis of PPi by inorganic pyrophosphatase makes these reactions practically irreversible. This hypothesis, which is now generally accepted, was stated more firmly by him in 1962.

PPi Concentration in Biological Material

Biosynthetic reactions produce large amounts of PPi, as shown by the calculations presented in the chapter 1.2. However, most authors of biochemical textbooks assume that very little of it exists in living cells, because it is hydrolysed immediately by PPase. This assumption is logical, for the removal of PPi prevents the seemingly futile loss of energy which would take place, if the biosynthetic reactions would go backwards in the presence of PPi. However, in the past fifty years PPi has been shown to exist throughout the living world. In this chapter I shall discuss these studies beginning from bacteria and going via lower eukaryotes and plants to animals and humans.

A method for concentration of nucleoside triphosphates by coprecipitation with calcium fluoride

A method for concentration of nucleoside triphosphates (NTP) is described. NTPs are quantitatively coprecipitated from the solution with calcium fluoride. The precipitate is separated by filtration through a membrane filter and NTPs are dissolved from the filter by immersing it in 0.5 N H2SO4. With this method also nucleoside diphosphates can be efficiently concentrated, but the method does not work with nucleoside monophosphates or cyclic AMP.

Regulatory Roles of PPi

In addition to being a source of biochemical energy PPi acts also as a regulator of biochemical reactions and processes. In many reports PPi has been proposed to: i. inhibit or activate some enzymes, ii. influencethe fidelity of protein or nucleic acid synthesis, iii. affect the formation of calcified tissues, cell proliferation and cellular iron transport, and iv. be involved in some pathologic conditions, formation of urinary stones and deposition of calcium pyrophosphate dihydrate crystals into knee joints (pseudogout) being the most studied examples. All these topics are discussed in this chapter.