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PubMed1981

Effect of colchicine on the Golgi apparatus and on GERL of rat jejunal absorptive cells. Ultrastructural localization of thiamine pyrophosphatase and acid phosphatase activity.

Ultrastructural localization of thiamine pyrophosphatase (TTP) and acid phosphatase (AcPase) activity was performed on jejunal absorptive cells of rats pretreated with the antimicrotubular agent colchicine and of control animals. Demonstration of TPP activity showed that most of the dislocated Golgi stacks after colchicine application lacked positively staining cisternae of the mature side. This cytochemical finding is in agreement with the morphologically demonstrable changes of the Golgi stacks resulting in a loss of polarity and give evidence for a colchicine-induced deficiency of the Golgi apparatus. The cytochemical localization of AcPase activity showed deposits of reaction product over lysosomes and GERL and demonstrated a dislocation of GERL occurring concomitantly with the changes of the Golgi apparatus. The antimicrotubular effect of colchicine is well documented; thus the morphological and cytochemical changes of the Golgi apparatus and of GERL might be due to a disturbed microtubular function after application of this agent suggesting an influence of microtubules in the maintenance of the integrity of these organelles. This hypothesis includes the possibility of an involvement of microtubules in formation and differentiation of Golgi stacks and GERL as well as a kind of "skeletal"function being responsible for their characteristic structure and fashion.

Acid Phosphatase↗
PubMed1981

[Stable compound of inorganic pyrophosphatase with pyrophosphate obtained by a fluoride-mediated reaction with phosphate].

Incubation of inorganic pyrophosphate from baker's yeast with phosphate and MgCl2 in the presence of fluoride results in a gradual inactivation of the enzyme concomitant with incorporation of PP1 (about 2 moles per mole) into the protein. The rate constant for this process shows an increase with a rise in concentrations of the three reagents, the maximal value of inactivation being 0.11 min-1. The bound PP1 is not separated by gel-filtration. The rate of spontaneous degradation of the enzyme-pyrophosphate complex and the nature of EDTA and Mg2+ effects are similar to those for the analogous compound obtained by inhibition of PP1 hydrolysis by fluoride. The data obtained suggest that during PP1 synthesis and hydrolysis by pyrophosphatase fluoride stabilizes the same intermediate of the enzyme with pyrophosphate.

Diphosphates↗
PubMed1982

[Calorimetric assay of yeast inorganic pyrophosphatase interaction with magnesium and phosphate ions].

The thermodynamic characteristics for the specific binding of one or two Mg2+ by the yeast inorganic pyrophosphatase and for the enzyme interaction with phosphate were determined. Saturation of the first binding site with Mg2+ causes structural rearrangements in the enzyme molecule without changing the temperature of protein denaturation. On the contrast, saturation of the second binding site results in stabilization of the system, i. e. a considerable fall in the entropy and a rise in the temperature of denaturation. Phosphorylation of the enzyme carboxylic group by inorganic phosphate requires saturation of the first binding site with Mg2+ and is not accompanied by changes in the enthalpy of the system. The pyrophosphate synthesis in the presence of the enzyme saturated with Mg2+ in both binding sites is associated with changes in the enthalpy and, possibly, in the entropy of the system.

Calorimetry↗
PubMed1982

[Some features of methylpyrophosphate hydrolysis by yeast inorganic pyrophosphatase].

The interaction of yeast inorganic pyrophosphatase with methylpyrophosphate was studied. In the presence of Mg2+ the rate of hydrolysis of the methylpyrophosphate-Zn2+ complex by the enzyme was shown to decrease. This was accompanied by competition of Zn2+ and Mg2+ for one site of Me2+ binding on the enzyme. The kinetics of combined hydrolysis of zinc methylpyrophosphate and zinc pyrophosphate were studied. It was found that both substrates are hydrolyzed at the same active site of the enzyme. Free methylpyrophosphate when bound to a specific phosphorylation site on the enzyme surface accelerated magnesium pyrophosphate hydrolysis. Some kinetic parameters of this hydrolysis were determined.

Binding Sites↗
PubMed1983

[Spatial distribution of thiamine pyrophosphatase and arylsulfatase activities in the area of the Golgi apparatus of the adenohypophyseal secretory cells in the white rat].

Specimens of albino rat pituitary glands were processed consecutively for demonstration of thiamine pyrophosphatase (TPPase) and aryl sulfatase (ArSase) activities. To differentiate between the structures associated with each particular enzyme studied within a single cell, either somatotroph or mammotroph, ultrathin sections were exposed for 2 minutes to 2--50% H2SO4 which removed the reaction products for TPPase rather than those for ArSase. The comparative study of pairs of micrographs of the same area taken before and after the etching with H2SO4 has shown TPPase and ArSase to reside in the Golgi apparatus and GERL system, respectively. Transitional elements have also been discovered, thus supporting the idea that GERL may be a derivative of the Golgi apparatus.

Animals↗
PubMed1983

[Submicroscopic study of adenohypophyseal cells with the hypophysis incubated in a hypertonic solution. I. The distribution of acid phosphatase, thiamine pyrophosphatase and arylsulfatase activities].

In the rat adenohypophyseal mammotrophs and somatotrophs, activities of acid phosphatase, thiamine pyrophosphatase and aryl sulphatase were defined after the incubation of the pituitary gland in the medium containing 1.65 M sucrose. Following 60--120 minutes of the incubation the above enzymes were found, in addition to their usual sites, in all cisternae of the Golgi apparatus and the rough endoplasmic reticulum as well as within the perinuclear cisternae. The mechanism of the above alterations is discussed, and a suggestion is put forward that the emergence of enzymatic activities in the above structures may reflect their biogenetic pathways in the cell.

Acid Phosphatase↗
PubMed1983

[Submicroscopic study of adenohypophyseal cells with the hypophysis incubated in a hypertonic solution. II. Differential analysis of the intracellular distribution of arylsulfatase and thiamine pyrophosphatase within a single cell].

In the rat pituitary gland exposed to an hour incubation in the medium containing 1.65 M sucrose activities of aryl sulphatase (AS) and thiamine pyrophosphatase (TPP) were successively defined within a single specimen. After the detection and registration of both enzyme activities with the electron microscope ultrathin sections were processed with 50% H2SO4 resulting in the complete removal of the reaction products for TPP. In the mammotrophs and somatotrophs, cisternae of the Golgi apparatus and rough endoplasmic reticulum showed either AS, or TTP or both the enzymatic activities. In the latter case the two enzymes could be restricted to a single cistern.

Animals↗
PubMed1984

[Membrane inorganic pyrophosphatase. "Syncatalytic" inactivation by the fluoride ion].

Two forms of inorganic pyrophosphatase isolated from the inner membrane and matrix of beef heart mitochondria as well as from the inner membrane of rat liver mitochondria lose their activity during incubation with NaF in the presence of a magnesium salt and PPi. The inactivation occurs in two steps. First, the F- ion combines with an enzyme-substrate complex in a readily reversible process with a dissociation constant of approximately 3 mM. Then, the ternary complex undergoes slow isomerization with a rate constant of 5 min-1. The half-time of the inhibitor dissociation from the complex is 8 min. The enzyme SH-group modification with maleimides or a disulfide enhances the affinity to F- at the first step. The F- ion incompetitively inhibits PPi hydrolysis. The mechanism of fluoride toxicity and the use of fluoride in the studies of PPi metabolism are discussed.

Animals↗
PubMed1994

The H(+)-pumping inorganic pyrophosphatase of the vacuolar membrane of higher plants.

The vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase) of plants is a member of a new class of energized ion translocases. The development of our understanding of this enzyme is briefly reviewed, including the evidence for its physiological role in H(+)-pumping and K+ transport into the vacuole, the identity of the polypeptides components, the cloning and sequencing of a cDNA encoding the catalytic subunit, and the partitioning of function between cytosolic and membrane domains of the protein. Lack of information about the identity of the substrate, activators and inhibitors of the H(+)-PPase has been a major barrier to the latter work. The various analyses that have been done of the kinetics of the enzyme predict different sets of activators and inhibitors, but work with residue-specific covalent inhibitors is now being done to resolve this. The results suggest that Mg2PPi is the substrate and that Mg2+ is an activator, but whether other PPi complexes inhibit the enzyme is still to be established.

Biological Transport, Active↗
PubMed1994

Circadian rhythmometric analysis of hepatic phosphohydrolases with special reference to glucose-6-phosphatase and inorganic pyrophosphatase.

Rhythmometric analysis of a group of phosphohydrolases in mouse liver has been performed along a single 24 hr time scale. The presence of the rhythm was conducted by F test. Statistically significant circadian rhythm was detected in glucose-6-phosphatase (G6Pase) and inorganic pyrophosphatase (InPPase) activity expressed on fresh weight and protein basis. Both G6Pase and InPPase oscillated with a high amplitude of 0.44 U and 1.15 U respectively across the mean value (mesor) of 0.40 +/- 0.42 U and 2.81 +/- 1.14 U per mg protein and with a phase shift of 80 degrees (5.34 hr) among them. On the other hand, alkaline phosphatase (AlPase) did not show any rhythm whereas adenosine triphosphatase (ATPase) showed rhythmic activity on protein basis and oscillated across mesor of 1.84 +/- 0.5 U with an amplitude of 0.52. Acrophase (time for peak activity/mg protein) of G6Pase, InPPase and ATPase was found at 194.2 degrees (13.34 hr), 114.1 degrees (8.0 hr) and at 306.1 degrees (20.4 hr) respectively. AlPase, though did not show significant rhythm, had peak value at 231.8 degrees. Since hepatic G6Pase is a multicomponent and multifunctional enzyme with several overlapping activities (viz. InPPase), coordinated action of G6Pase and InPPase in the regulation of hepatic cell functions has been suggested.

Animals↗
PubMed1994

Localization of cytosolically oriented maleimide-reactive domain of vacuolar H(+)-pyrophosphatase.

The vacuolar H(+)-pyrophosphatase (V-PPase) of plant cells is subject to substrate (Mg2PPi)-protectable, free PPi-potentiated irreversible inhibition by the sulfhydryl reagent N-ethylmaleimide (NEM). Inhibition by NEM approximates pseudo-first order kinetics and double-log plots of the first order rate constant for inactivation versus NEM concentration yield a straight line relationship with a slope of approximately unity. Since NEM and the membrane-impermeant cysteine reagent 3-(N-maleimidylpropionyl)biocytin (MPB) inhibit the V-PPase with similar kinetics and compete for a common binding site on the M(r) = 66,000 substrate-binding subunit, a single residue located in a cytosolically disposed extramembranous domain is inferred to undergo covalent modification in both cases. Selective labeling of the V-PPase of vacuolar membrane vesicles with [14C]NEM, purification of the M(r) = 66,000 subunit, and its digestion with V8 protease generates multiple peptide fragments. Of the bands identified after electrophoresis of the digests on Tris-Tricine gels, only one, migrating at M(r) = 14,000 (V814K), contains 14C label. Gas-phase sequence analysis of this band after electrotransfer to Immobilon PSQ yields two overlapping sequences (V814K2 and V814K2) which unambiguously align with the carboxyl-terminal segment of the M(r) = 66,000 subunit. Both V814K1 and V814K2 encompass only 1 cysteine residue at position 634 which is conserved between the V-PPases from Arabidopsis thaliana, Beta vulgaris (isoforms 1 and 2), and Hordeum vulgare. On the basis of these findings, the strict conservation of the sequence of the V-PPase from multiple plant sources, and the identical kinetics of interaction of the enzymes from Vigna and Beta with NEM and MPB, Cys634 of putative hydrophilic loop X is concluded to be the cytosolically oriented residue whose alkylation by maleimides is responsible for inactivation of the V-PPase. The significance of these results with respect to earlier speculations concerning the identity of the catalytic site and topology of the V-PPase is discussed.

Amino Acid Sequence↗
PubMed1993

Amino acid sequence similarities between the vacuolar proton-pumping inorganic pyrophosphatase and the c-subunit of F0F1-ATPases.

Comparison of the Arabidopsis thaliana vacuolar proton-pumping inorganic pyrophosphatase with three F0F1-ATPase c-subunits revealed a strong similarity between a stretch containing amino acids 227-245 of the H(+)-PPase and a transmembrane alpha-helix of the c-subunits which contains the glutamate which binds N,N'-dicyclohexylcarbodiimide.

Amino Acid Sequence↗
PubMed1996

Purification and kinetic characterization of chicken liver inorganic pyrophosphatase.

Soluble inorganic pyrophosphatase (EC 3.6.1.1) was isolated from chicken liver, RIR breed, to apparent homogeneity. The enzyme showed a molecular mass of 100 kDa as estimated by gel filtration and a subunit mass of 49 kDa on SDS-PAGE. The enzyme was very specific for pyrophosphate (PPi) and magnesium, and there was no measurable activity on replacing Mg2+ with Zn2+. At optimal conditions of assay, 50% of the enzyme activity was inhibited at 42 microM Ca2+, 70 microM fluoride and 0.91 mM Cd2+. There was a 50% inactivation of enzyme activity at 0.1 M guanidine hydrochloride (GuHCl). Kinetic analysis of GuHCl inactivation revealed 2 essential binding sites for this ligand. The enzyme showed allosteric behaviour with the substrate PPi and Mg2+. The apparent Hill coefficient of 1.47 and 1.48 for PPi and Mg2+, respectively indicate positive cooperatively. Hill plots also gave [S]0.5 of 0.177 mM and 2.5 microM for Mg2+ and PPi, respectively.

Animals↗
PubMed1997

[Irreversible specific inhibition of E. coli inorganic pyrophosphatase with amines].

An unusually high reactivity of the carboxyl groups of the active site of E. coli inorganic pyrophosphatase towards amines was shown. Amino acid esters and other amines are specific irreversible inhibitors of the enzyme. The reaction involves the formation of an enzyme-inhibitor complex followed by the chemical modification of dicarboxylic amino acid residues. It is assumed that the binding of the positively charged inhibitor occurs at the binding site of cations-activators.

Amines↗
PubMed1997

Effect of Asp-97-->Glu substitution on the pH dependence of catalysis by inorganic pyrophosphatase of Escherichia coli.

Substitution of Glu for the evolutionarily conserved Asp-97 in the active site of Escherichia coli inorganic pyrophosphatase increases the apparent pKa of the essential acidic group controlling the catalytic constant for pyrophosphate hydrolysis. In combination with previously reported data, this fact suggests that activity decreases in alkaline medium because of decreased rate of the release of the first molecule of the product phosphate from the active site.

Amino Acid Substitution↗
PubMed1998

Changes in E. coli inorganic pyrophosphatase structure induced by binding of metal activators.

The three-dimensional structures of E. coli inorganic pyrophosphatase (PPase) and its complexes with Mn2+ in a high affinity site and with Mg2+ in high and low affinity sites determined by authors in 1994-1996 at 1.9-2.2 A resolution are compared. Metal ion binding initiates the shifts of alpha-carbon atoms and of functional groups and rearrangement of non-covalent interaction system of hexameric enzyme molecule. As a result, the apoPPase with six equal subunits turns after Mg2+ binding into the structure with three types of subunits distinguished by structure and occupance of the low affinity Mg2+ site. Induced asymmetry reflects the subunit interactions and cooperativity between Mg2+ binding sites. These molecular rearrangements are structural basis to account for special features of the enzyme behavior and to propose one of the pathways for enzymatic activity regulation of constitutive PPases in vivo.

Apoenzymes↗
PubMed1998

Three-dimensional structures of mutant forms of E. coli inorganic pyrophosphatase with Asp-->Asn single substitution in positions 42, 65, 70, and 97.

The three-dimensional structures of four mutant E. coli inorganic pyrophosphatases (PPases) with single Asp-->Asn substitutions at positions 42, 65, 70, and 97 were solved at 1.95, 2.15, 2.10, and 2.20 A resolution, respectively. Asp-42-->Asn and Asp-65-->Asn mutant PPases were prepared as complexes with sulfate--a structural analog of phosphate, the product of enzymatic reaction. A comparison of mutant enzymes with native PPases revealed that a single amino acid substitution changes the position of the mutated residue as well as the positions of several functional groups and some parts of a polypeptide chain. These changes are responsible for the fact that mutant PPases differ from the native ones in their catalytic properties. The sulfate binding to the mutant PPase active site causes molecular asymmetry, as shown for the native PPase earlier. The subunit asymmetry is manifested in different positions of sulfate and several functional groups, as well as changes in packing of hexamers in crystals and in cell parameters.

Amino Acid Substitution↗