THE METABOLISM OF THE ERYTHROCYTE: X. THE INORGANIC PYROPHOSPHATASE OF THE ERYTHROCYTE

1956 ◽  
Vol 34 (2) ◽  
pp. 121-129 ◽  
Author(s):  
A. Malkin ◽  
O. F. Denstedt
Keyword(s):  
Enzyme Activity ◽  
Calcium Ions ◽  
Human Erythrocyte ◽  
Inorganic Pyrophosphatase ◽  
Magnesium Ions ◽  
Constant Ratio ◽  
Inorganic Pyrophosphate ◽  
Noncompetitive Inhibitor ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of

The activity of the pyrophosphatase which catalyzes the hydrolysis of inorganic pyrophosphate in the erythrocyte of the human, the rabbit, and the chicken is confined entirely to the cytoplasm of the cell. Following preincubation, the enzyme activity in the human erythrocyte is diminished, but pre-incubation in the presence of cysteine or glutathione prevents the diminution of the enzyme activity. Aging of the hemolyzate of the human erythrocytes results in a marked loss of the inorganic pyrophosphatase activity. The diminished activity can be restored by the addition of cysteine or glutathione to the reaction mixture; but after the hemolyzate has aged for five or six days at 5 °C, the loss in the enzyme activity can no longer be restored with these reagents. Fluoride and calcium ions inhibit the activity of the enzyme, while magnesium ions are essential for its activity. Calcium is a noncompetitive inhibitor, while the inhibition by fluoride is of a "quadratic" nature. If a constant ratio of magnesium to pyrophosphate is maintained, the quadratic inhibition can be converted to the "uncompetitive" type of inhibition.

THE METABOLISM OF THE ERYTHROCYTE: X. THE INORGANIC PYROPHOSPHATASE OF THE ERYTHROCYTE

1956 ◽  
Vol 34 (1) ◽  
pp. 121-129 ◽  
Author(s):  
A. Malkin ◽  
O. F. Denstedt
Keyword(s):  
Enzyme Activity ◽  
Calcium Ions ◽  
Human Erythrocyte ◽  
Inorganic Pyrophosphatase ◽  
Magnesium Ions ◽  
Constant Ratio ◽  
Inorganic Pyrophosphate ◽  
Noncompetitive Inhibitor ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of

The activity of the pyrophosphatase which catalyzes the hydrolysis of inorganic pyrophosphate in the erythrocyte of the human, the rabbit, and the chicken is confined entirely to the cytoplasm of the cell. Following preincubation, the enzyme activity in the human erythrocyte is diminished, but pre-incubation in the presence of cysteine or glutathione prevents the diminution of the enzyme activity. Aging of the hemolyzate of the human erythrocytes results in a marked loss of the inorganic pyrophosphatase activity. The diminished activity can be restored by the addition of cysteine or glutathione to the reaction mixture; but after the hemolyzate has aged for five or six days at 5 °C, the loss in the enzyme activity can no longer be restored with these reagents. Fluoride and calcium ions inhibit the activity of the enzyme, while magnesium ions are essential for its activity. Calcium is a noncompetitive inhibitor, while the inhibition by fluoride is of a "quadratic" nature. If a constant ratio of magnesium to pyrophosphate is maintained, the quadratic inhibition can be converted to the "uncompetitive" type of inhibition.

Inorganic pyrophosphatase activity in oral streptococci: purification and properties of the enzyme from Streptococcus salivarius

1982 ◽  
Vol 60 (4) ◽  
pp. 452-462 ◽  
Author(s):  
Ramji L. Khandelwal ◽  
Ian R. Hamilton
Keyword(s):  
Enzyme Activity ◽  
Deae Cellulose ◽  
Inorganic Pyrophosphatase ◽  
Phosphate Ester ◽  
Oral Streptococci ◽  
Streptococcus Salivarius ◽  
Inorganic Pyrophosphate ◽  
Magnesium Pyrophosphate ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of

Inorganic pyrophosphatase has been purified from the soluble fraction of Streptococcus salivarius by protamine sulfate treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was purified approximately 500-fold with a 33% yield. The purified enzyme was homogeneous since it showed a single band when examined by nondenaturing polyacrylamide gel electrophoresis. It was rich in acidic (glutamic and aspartic) amino acids, as well as serine and glycine. The enzyme was devoid of sulfur-containing amino acids.The purified enzyme was specific for the hydrolysis of inorganic pyrophosphate and did not hydrolyze any other phosphate-ester compound examined. Inorganic pyrophosphatase activity was completely dependent on a divalent cation. Activity was maximum in the presence of Mg2+ while activity in the presence of Mn2+ and Co2+ was significantly lower. In the presence of Mg2+, a number of divalent cations, however, inhibited the enzyme activity. The true substrates for S. salivarius inorganic pyrophosphatase were magnesium–pyrophosphate complexes, i.e., MgPPi and Mg2PPi, while free Mg2+ had no effect on the enzyme activity and free PPi inhibited the hydrolysis of inorganic pyrophosphate. Km value for magnesium–pyrophosphate complexes was 16.4 μM. Km value for total Mg2+ was similar ranging between 14.4 and 20 μM. Analysis of data by Hill plots indicated one binding site for Mg2+ and two for PPi. Among various nucleotides and glycolytic intermediates examined, GDP, GMP, and fructose-1,6-P2 showed significant inhibitory effect on enzyme activity.

scholarly journals CRYSTALLINE INORGANIC PYROPHOSPHATASE ISOLATED FROM BAKER'S YEAST

1952 ◽  
Vol 35 (3) ◽  
pp. 423-450 ◽  
Author(s):  
M. Kunitz
Keyword(s):  
Molecular Weight ◽  
Enzymatic Hydrolysis ◽  
Isoelectric Point ◽  
Calcium Ions ◽  
Baker’S Yeast ◽  
Thiamine Pyrophosphate ◽  
Inorganic Pyrophosphatase ◽  
Baker's Yeast ◽  
Inorganic Pyrophosphate ◽  
Hydrolysis Of

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40°C. and pH 7.0 at a concentration of 3 to 4 x 10–3 M Na4P2O7 and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na4P2O7 or K4P2O7 proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme.

THE HYDROLYSIS OF MONOPHOSPHOINOSITIDE BY EXTRACTS OF BRAIN

1967 ◽  
Vol 45 (6) ◽  
pp. 853-861 ◽  
Author(s):  
W. Thompson
Keyword(s):  
Fatty Acids ◽  
Enzyme Activity ◽  
Calcium Ions ◽  
Brain Extract ◽  
Monovalent Cations ◽  
High Concentration ◽  
Diffusible Factor ◽  
Hydrolysis Of ◽  
The Brain ◽  
Long Chain

The hydrolysis of monophosphoinositide by soluble extracts from rat brain is described. Diglyceride and inositol monophosphate are liberated along with a small amount of free fatty acids. Hydrolysis of the lipid is optimal at pH 5.4 in acetate buffer. The reaction is stimulated by calcium ions or by high concentration of monovalent cations and, to a less extent, by long-chain cationic amphipathic compounds. Enzyme activity is lost on dialysis of the brain extract and can be restored by diffusible factor(s). Some differences in the conditions for hydrolysis of mono- and tri-phosphoinositides are noted.

scholarly journals Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1211-C1211
Author(s):  
Joseph Ng ◽  
Ronny Hughes ◽  
Michelle Morris ◽  
Leighton Coates ◽  
Matthew Blakeley ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Crystallographic Structures ◽  
Hydrolysis Of ◽  
Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

scholarly journals Effects of spermine and spermidine on the inorganic pyrophosphatase of Streptococcus faecalis. Interactions between polyamines and inorganic pyrophosphate

1989 ◽  
Vol 259 (1) ◽  
pp. 55-59 ◽  
Author(s):  
R Lahti ◽  
R Hannukainen ◽  
H Lönnberg
Keyword(s):  
Gamma Ray ◽  
Inorganic Pyrophosphatase ◽  
Emission Analysis ◽  
Streptococcus Faecalis ◽  
Inorganic Pyrophosphate ◽  
Bivalent Cations ◽  
Hydrolysis Of ◽  
Apparent Stability ◽  
Allosteric Activator

We have shown a dual role for Mg2+ in the hydrolysis of PPi catalysed by inorganic pyrophosphatase (PPase; EC 3.6.1.1) of Streptococcus faecalis; Mg2+ is necessary for the formation of the substrates, Mg1PPi2- and Mg2PPi0, and it also acts as an allosteric activator [Lahti + Jokinen (1985) Biochemistry 24, 3526-3530]. No activity can be observed with S. faecalis PPase in the absence of bivalent cations, which indicates that free PPi cannot serve as a substrate for this enzyme. However, significant activities were observed in the presence of spermine and spermidine, even though no bivalent cations were present. It was shown by particle-induced gamma-ray emission and particle-induced X-ray-emission analysis that the polyamines used were not contaminated with Mg2+ or any other bivalent cations that could support PPase activity. Hence it is obvious that polyamines are able to form a complex with PPi that serves as a substrate for PPase. The apparent stability constants for the 1:1 adducts of spermine and spermidine were estimated by a resin competition method. The values obtained at pH 7.5 were 2.7 X 10(3) M-1 and 6.4 X 10(2) M-1 respectively. Kinetic results further suggested that polyamines can also substitute for Mg2+ as an activator in vitro. The physiological significance of these polyamine effects were discussed.

scholarly journals Hydrolysis of Adenosine Triphosphate by Crystalline Yeast Pyrophosphatase

1962 ◽  
Vol 45 (4) ◽  
pp. 31-46 ◽  
Author(s):  
M. Kunitz
Keyword(s):  
Ion Exchange ◽  
Protein Molecule ◽  
Molar Ratio ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Zn Ions ◽  
Dowex 1

Schlesinger and Coon's report that crystalline yeast inorganic pyrophosphatase, in addition to its known ability to hydrolyze inorganic pyrophosphate in the presence of Mg ions, is also able to catalyze the hydrolysis of ATP and ADP in the presence of Zn ions was confirmed. A systematic study showed that the ratio of 370 of PPase-Mg over ATPase-Zn activities per milligram protein in various preparations of pyrophosphatase obtained in the course of isolation of crystalline pyrophosphatase from baker's yeast was nearly identical in all the preparations, independent of their purity. The course of hydrolysis of ATP by crystalline pyrophosphatase in the presence of Zn was carried out with the aid of ion exchange on Dowex 1. The finding of Schlesinger and Coon that the hydrolysis proceeds from ATP to ADP and then slowly to AMP was confirmed. The kinetics of the first phase of the reaction was found to depend on the molar ratio of Zn/ATP in the reaction mixture. Mg ions in the presence of Zn ions have an accelerating effect on the rate of hydrolysis of ATP. This suggests strongly that both activities—ATPase and PPase—are manifestations of the same active group in the protein molecule of crystalline pyrophosphatase.

scholarly journals The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1969 ◽  
Vol 112 (5) ◽  
pp. 699-701 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Competitive Inhibitor ◽  
Inorganic Pyrophosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Low Concentrations ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of ◽  
Pyrophosphate Hydrolysis

1. The differential effects of adding Zn2+ and Mg2+ on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn2+, inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn2+–pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg2+ also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.

scholarly journals Biological properties and roles of a Trichinella spiralis inorganic pyrophosphatase in molting and developmental process of intestinal larval stages

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Chen Xi Hu ◽  
Jie Zeng ◽  
Hui Nan Hao ◽  
Yang Xiu Yue Xu ◽  
Fang Liu ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Metal Binding ◽  
Developmental Process ◽  
Trichinella Spiralis ◽  
Biological Properties ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
Larval Stages ◽  
Molting Process ◽  
Hydrolysis Of

AbstractInorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg2+, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.

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