Inorganic Pyrophosphatase in Storage Buffer

2013 ◽  
Vol 2013 (11) ◽  
pp. pdb.rec079392-pdb.rec079392
Keyword(s):  
Inorganic Pyrophosphatase ◽  
Storage Buffer

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

EFFECT OF THYROCALCITONIN ON ALKALINE PHOSPHATASE AND PYROPHOSPHATASE IN RAT TIBIA, KIDNEY AND INTESTINE

1972 ◽  
Vol 70 (4) ◽  
pp. 676-682 ◽  
Author(s):  
Cipora Streifler ◽  
Arie Orenstein ◽  
Arieh Harell
Keyword(s):  
Alkaline Phosphatase ◽  
Kidney Tissue ◽  
Plasma Calcium ◽  
Inorganic Pyrophosphatase ◽  
Rat Tibia ◽  
Rat Bone

ABSTRACT The influence of thyrocalcitonin (TCT) on the enzymes alkaline phosphatase and inorganic pyrophosphatase in rat bone, kidney and intestine was studied. The rats were injected with TCT every hour for 4 hours. They were divided into groups and were sacrificed 1 h after the first, second, third and fourth injection respectively. The plasma calcium was found to be reduced. Enzyme studies showed that: a) in tibia metaphysis homogenates alkaline phosphatase increased in response to TCT, to 198, 175, 154 and 183 per cent of the non-injected rats after 1, 2, 3, and 4 injections, respectively; inorganic pyrophosphatase was elevated to 356, 209, 221, 425 per cent after the same TCT injections. b) In kidney homogenates alkaline phosphatase was reduced to 75, 53, 79, 68 per cent of the non-injected rats after 1, 2, 3 and four doses, respectively; inorganic pyrophosphatase was reduced to 78, 56, 77 and 71 per cent after the same injections of TCT. c) In the jejunum, alkaline phosphatase was found to be 88.5, 71, 91 and 115 per cent of the untreated rats after 1, 2, 3 and 4 injections, respectively; pyrophosphatase in this tissue was found to be 105, 102, 102 and 113 per cent of the normal under the above conditions. The results indicate: 1. TCT causes increases in alkaline phosphatase and inorganic pyrophosphatase activities in bone. The increase of pyrophosphatase is significantly more marked than the increase of alkaline phosphatase; 2. in kidney tissue, the action of TCT on these two enzymes is slower and their activities are equally reduced; 3. in the jejunum no significant effect of TCT on the activity of these two enzymes was observed.

scholarly journals Identification and Characterization of Inorganic Pyrophosphatase and PAP Phosphatase from Thermococcus onnurineus NA1

2009 ◽  
Vol 191 (10) ◽  
pp. 3415-3419 ◽  
Author(s):  
Hyun Sook Lee ◽  
Yun Jae Kim ◽  
Jung-Hyun Lee ◽  
Sung Gyun Kang
Keyword(s):  
Gene Clusters ◽  
Inorganic Pyrophosphatase ◽  
Thermococcus Onnurineus Na1 ◽  
First Report ◽  
Activation System ◽  
Sulfate Activation ◽  
Pyrococcus Abyssi ◽  
Identification And Characterization ◽  
Hypothetical Genes

ABSTRACT Two hypothetical genes were functionally verified to be a pyrophosphatase and a PAP phosphatase in Thermococcus onnurineus NA1. This is the first report of the pyrophosphatases and the PAP phosphatases being organized in the gene clusters of the sulfate activation system only in T. onnurineus NA1 and “Pyrococcus abyssi.”

scholarly journals Constitutive Inorganic Pyrophosphatase of Escherichia coli

1970 ◽  
Vol 245 (17) ◽  
pp. 4358-4364 ◽  
Author(s):  
Pamela M. Burton ◽  
John Josse
Keyword(s):  
Escherichia Coli ◽  
Inorganic Pyrophosphatase

scholarly journals Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

1975 ◽  
Vol 23 (5) ◽  
pp. 342-347 ◽  
Author(s):  
A Linde ◽  
B C Magnusson
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Adenosine Triphosphatase ◽  
Ruthenium Red ◽  
Inorganic Pyrophosphatase ◽  
Alkaline Ph ◽  
Hard Tissue ◽  
Assay Condition ◽  
Phosphatase Inhibitors ◽  
Inhibition Studies

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

2010 ◽  
Vol 43 (5) ◽  
pp. 1113-1120 ◽  
Author(s):  
Esko Oksanen ◽  
François Dauvergne ◽  
Adrian Goldman ◽  
Monika Budayova-Spano
Keyword(s):  
Data Collection ◽  
Neutron Diffraction ◽  
Diffraction Data ◽  
Catalytic Mechanism ◽  
Inorganic Pyrophosphatase ◽  
Neutron Diffraction Data ◽  
Cooling Device ◽  
Data Set ◽  
X Ray ◽  
X Ray Crystallography

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

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