Isolation and primary characterization of two forms of inorganic pyrophosphatase from ox heart muscle mitochondria

A method allowing to obtain two highly purified forms (membrane-bound and soluble ones) of inorganic pyrophosphatase from bovine heart mitochondria is described. Both forms have the isoelectric point of 5.8, pH optimum with Mg2+ at 7--9, are maximally stable at pH 5.8 and absolutely specific to PPi and require Mg2+ or Co2+ for their activity. The soluble pyrophosphatase is also activated by Zn2+. Besides, the two forms differ in their electrophoretic mobilities, affinity for DEAE cellulose and stability in acidic (pH less than 5.8) and alkaline media.     

Catalytic properties of inorganic pyrophosphatase in rat liver mitochondria]

Intact rat liver mitochondria possess a very low hydrolytic activity, if any, towards exogenous pyrophosphate. This activity can be unmasked by making mitochondria permeable to PPi by toluene treatment or by disrupting them with detergents or ultrasound, thus indicating that the active site of pyrophosphatase is localized in the matrix. The initial rates of PPi hydrolysis of toluene-permeabilized mitochondria and purified pyrophosphatase were found to depend, in a similar manner, on PPi and Mg2+ concentrations. The simplest model consistent with these data in both cases implies that the reaction proceeds via two pathways and requires MgPPi as substrate and at least one Mg2+ ion as activator. In the presence of 0.4 mM Mg2+ (physiological concentration) the inhibition constant for Ca2+ is 12 microM and the enzyme activity is no less than 50% of the maximal one. The data obtained suggest that the activity of pyrophosphatase in mitochondria is high enough to keep free PPi concentration at a level close to the equilibrium one.     

Catalytic properties of the inorganic pyrophosphatase in rat liver mitochondria.

Intact rat liver mitochondria have very low hydrolytic activity, if any, toward exogenous pyrophosphate. The activity can be unmasked by making mitochondria permeable to PPi by toluene treatment or disrupting them with detergents or ultrasound, indicating that the active site of pyrophosphatase is located in the matrix. Initial rates of PPi hydrolysis by toluene-permeabilized mitochondria and purified pyrophosphatase were found to depend in a similar manner on PPi and Mg2+ concentrations. The simplest model consistent with the data in both cases implies that the reaction proceeds through two pathways and requires MgPPi as the substrate and, at least, one Mg2+ ion as the activator. In the presence of 0.4 mM Mg2+ (physiological concentration), the inhibition constant for Ca2+ is 12 microM and the enzyme activity is, at least, 50% maximal. The results suggest that the activity of pyrophosphatase in mitochondria is high enough to keep free PPi concentration at a level close to that at equilibrium.     

Inhibition of inorganic pyrophosphatase of animal mitochondria by calcium

Calcium ion is an uncompetitive inhibitor of the inorganic pyrophosphatases of bovine heart and rat liver mitochondria with respect to substrate MgPPi at pH 8.5 and a non-competitive inhibitor of the former enzyme at pH 7.2. The concentration of Ca2+ required to decrease the maximal velocities for both enzymes at pH 8.5, 0.4 mM Mg2+ was about 10 microM. The inhibition results from the binding of two Ca2+ ions to both free enzymes and their complexes with the substrate. The results suggest that Ca2+ regulates pyrophosphatase activity and hence PPi level in mammalian mitochondria.     

Subcellular localization of inorganic pyrophosphatase in rat salivary glands

Inorganic pyrophosphatase (EC 3.6.1.1) from cells of the sublingual and submandibular salivary glands of rat was found only in the cytosol and was absent in nuclei, mitochondria, lysosomes and microsomes.     

Purification and subunit structure of phenylalanyl-tRNA synthetase from hen liver mitochondria

Hen liver mitochondrial phenylalanyl-tRNA synthetase is purified to homogeneity by a series of steps including salting-out chromatography, salting-out affinity chromatography in the presence of tRNA^Phe, dissociation of the enzyme-tRNA complex on DEAE-cellulose, chromatography on DEAE-Sepharose CL-6B and Sepharose 6B. The enzyme appears to be a tetrameric enzyme with a molecular weight of 255 000, as determined by gel filtration, with a subunit structure of α₂β₂ (α = 57 000, β = 66 000), as determined by sodium dodecyl sulfate gel electrophoresis.     

Kinetic studies on the interactions of two forms of inorganic pyrophosphatase of heart mitochondria with physiological ligands

A scheme of interactions of Mg2+ ions and their 1:1 complex with PPi (PPiMg') with two forms of inorganic pyrophosphatase isolated from beef heart mitochondria has been deduced from the analysis of enzyme kinetics at pH varying from 5.6 to 8.5. The scheme implies the existence of two catalytically important metal-binding sites on the enzyme. The two enzyme forms differ in maximal velocity and affinity for the metal activator. The pH dependence of kinetic parameters suggests that the active form of the substrate is MgP2O2-7. Ca2+ ions strongly inhibit pyrophosphatase activity and the corresponding Hill coefficient is 1.5. Phosphate and ATP are weak inhibitors of pyrophosphatase of the competitive and noncompetitive type respectively. The results show that these forms of mitochondrial pyrophosphatase are similar to pyrophosphatases isolated from other sources.     

Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase.

The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 A. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 A. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion. The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 A/Calpha. But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.     

Submitochondrial localization and function of alkaline inorganic pyrophosphatase in maize seedlings.

Alkaline inorganic pyrophosphatase and Mg-ATPase are localized within the mitoplast of maize seeding mitochondria. NaF inhibited the PPase activity, whereas oligomycin and dicyclohexylcarbodiimide inhibited the Mg-ATPase activity. The mitoplast preparation synthesized PPi from Pi under conditions excluding hydrolysis of endogenous ATP. PPi synthesis was inhibited by ADP, antimycin A, NaCN and 2,4- dinitrophenol but not by oligomycin. It is suggested that PPi synthesis in the maize seedling mitochondria proceeds at the expense of the energy of electron transport chain and is independent of the ATP synthesis.     

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.     

Inorganic pyrophosphatase of rat liver cytosol. Isolation and properties

An electrophoretically homogeneous preparation of rat liver cytosolic pyrophosphatase was obtained by a combination of chromatographic methods. The enzyme molecule is made up of two identical subunits, each about 38 kD. The enzyme is activated by Mg2+ and strongly inhibited by Ca2+. Both cations are bound on a time scale of minutes. Ca2+ binding occurs in two steps. EGTA-like metal chelators activate pyrophosphatase, presumably due to the removal of trace amounts of Ca2+ from solutions.