Enzymatic reactions involving orthoarsenate: arsenate is competitive with sulfate in the ATP sulfurylase reaction

In the presence of ATP, Mg2+, and arsenate, ATP sulfurylase from yeast will catalyze the formation of inorganic pyrophosphate. Inorganic pyrophosphate was detected by determination of orthophosphate in the presence of inorganic pyrophosphatase. Two moles of Pi were found for each molecule of ATP in the reaction mixture. The activity of ATP sulfurylase with arsenate as an activating anion was from 1 to 3% of the activity observed with molybdate.     

Colorimetric determination of orthophosphate in the assay of inorganic pyrophosphatase activity

Using Baginski and Zak's (12) method for determining inorganic orthophosphate as a starting point, a number of conditions which influence the accuracy and precision of the determination of pyrophosphatase activity have been shown: nonenzyme-catalyzed acid hydrolysis by protein precipitation agents and molybdate-catalyzed hydrolysis of pyrophosphate together with interference in the determination of orthophosphate by the substrate pyrophosphate and other components from the reaction mixture, Tris, chloride, acetate, citrate, boric acid. With regard to these sources of error, a method is described for determining pyrophosphatase activity, and its reliability is investigated.     

Hydrolysis of Inorganic Pyrophosphate in Dictyostelium discoideum

The hydrolysis of inorganic pyrophosphate has been studied in cell-free extracts prepared at different stages of development of Dictyostelium discoideum. Two enzyme reactions, pH optima 7.25 and 9.0, appear specific for inorganic pyrophosphate and have an absolute requirement for a divalent cation, preferably Mg²⁺. The enzyme specific activities do not change significantly during differentiation. Neither enzyme reaction is inhibited by orthophosphate and the presence of exogenous potassium phosphate does not affect the levels of pyrophosphalase at any stage. Exogenous glucose raises the pyrophosphatase levels in the sorocarps.     

Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores

Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing P(i) as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg(2+) concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores.     

Urinary Pyrophosphate in Normal Subjects and in Stone Formers

The urinary excretion of inorganic pyrophosphate was determined in nine normal subjects and also in eight patients with recurrent calcium-containing renal stones during varied levels of phosphate intake. The excretion of pyrophosphate and orthophosphate is virtually the same in the two groups at all levels of phosphate intake. It appears unlikely that a consistently reduced urinary excretion of pyrophosphate is a factor in the formation of urinary calculi. Pyrophosphate excretion rose and calcium excretion fell with increasing phosphate intake; this might be expected to have a beneficial effect in patients with recurrent calcium stones.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - PPi inorganic pyrophosphate - PGA 3-phosphoglycerate - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(aminoethyl ether) - N,N tetraacetic acid     

Chemical Conversion of Ribonucleoside 5′-S-Methyl Phosphorothiolates into Ribonucleotide Anhydrides Including Adenosine 5′-Triphosphate and Uridine Diphosphate Glucose

Ribonucleotide anhydrides have been prepared from corresponding ribonucleoside 5′-S-methyl phosphorothiolates by demethylthiolation with iodine in dry pyridine at room temperature in the presence of appropriate phosphates such as inorganic orthophosphate, inorganic pyrophosphate or glucose 1-phosphate. Thus synthesis of ribonucleotide anhydrides have been achieved and three ribonucleoside 5′-triphosphates (ATP, CTP and UTP), three ribonucleoside 5′-diphosphates (ADP, CDP and UDP) and a pyrophosphate coenzyme (UDPG) have been synthesized and isolated as lithium salts by charcoal treatment followed by ion exchange chromatography.     

Some effects of inorganic phosphate on O2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O₂ in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.

2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O₂ were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.

3. With CO₂ as the sole added substrate the extent of the initial lag in O₂ evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO₂.

4. There was also a requirement for phosphate by chloroplasts evolving O₂ in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO₂. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O₂ evolution which normally followed the addition of 3-phosphoglycerate.

5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.     

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

1. The differential effects of adding Zn(2+) and Mg(2+) on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn(2+), inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn(2+)-pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg(2+) 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.     

Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones?

An increasing body of biochemical and genetic evidence suggests that inorganic pyrophosphate (PPi) plays an important role in protist bioenergetics. In these organisms, two types of inorganic pyrophosphatases [EC 3.6.1.1, namely soluble PPases (sPPases) and proton-translocating PPases (H+-PPases)] that hydrolyse the PPi generated by cell anabolism, thereby replenishing the orthophosphate pool needed for phosphorylation reactions, are present in different cellular compartments. Photosynthetic and heterotrophic protists possess sPPases located in cellular organelles (plastids and mitochondria), where many anabolic and biosynthetic reactions take place, in addition to H+-PPases, which are integral membrane proteins of the vacuolysosomal membranes and use the chemical energy of PPi to generate an electrochemical proton gradient useful in cell bioenergetics. This last category of proton pumps was considered to be restricted to higher plants and some primitive photosynthetic bacteria, but it has been found recently in many protists (microalgae and protozoa) and bacteria, thus indicating that H+-PPases are much more widespread than previously thought. No cytosolic sPPase (in bacteria, fungi and animal cells) has been shown to occur in these lower eukaryotes. The widespread occurrence of these key enzymes of PPi metabolism among evolutionarily divergent protists strongly supports the ancestral character of the bioenergetics based on this simple energy-rich compound, which may play an important role in survival under different biotic and abiotic stress conditions.     

Gene ytkD of Bacillus subtilis encodes an atypical nucleoside triphosphatase member of the Nudix hydrolase superfamily

Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli. The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates. Whereas all other nucleoside triphosphatase members of the superfamily release inorganic pyrophosphate and the cognate nucleoside monophosphate, YtkD hydrolyses nucleoside triphosphates in a stepwise fashion through the diphosphate to the monophosphate, releasing two molecules of inorganic orthophosphate. Contrary to a previous report, our enzymological and genetic studies indicate that ytkD is not an orthologue of E. coli mutT.     

Pyrophosphate of high and low energy. Contributions of pH, Ca2+, Mg2+, and water to free energy of hydrolysis

The equilibrium between inorganic pyrophosphate and inorganic orthophosphate was determined at pH values varying between 6.0 and 8.0, in the presence of different concentrations of MgCl2, mixtures of MgCl2 and CaCl2, and different organic solvents. The reactions were catalyzed by yeast inorganic pyrophosphatase. It was found that at 35 degrees C, depending on the conditions used, the observed equilibrium constant of pyrophosphate hydrolysis vary from a value higher than 4 X 10(3) M (delta Goobs more negative than -5.1 kcal/mol) to a value as low as 3 M (delta Goobs -0.7 kcal/mol). The experimental data were used to compute the equilibrium constants of the reactions involving different ionic species. The data presented are interpreted according to the concept that the Keq of hydrolysis of a high energy compound depends on the difference in solvation energy of reactants and products.     

蒙东地区水肥耦合对紫花苜蓿土壤磷组分的影响

磷素在土壤中可分为有机磷和无机磷两大组分,不同形态的磷供给植物营养的难易程度不同,应用液体³¹P核磁共振技术(³¹P-NMR)探明土壤磷组分可为进一步调控土壤磷营养提供重要的理论依据。本研究采用盆栽试验,以紫花苜蓿和栗钙土为对象,设置常规和干旱水分处理,并设置不同的施磷水平(P₀～P₄:0、0.025、0.05、0.1、0.2 g P₂O₅·kg^-1土),应用液体³¹P-NMR技术测定土壤磷组分,研究水肥耦合条件下紫花苜蓿土壤磷组分特征。结果表明: 不同水肥条件下,土壤无机磷主要包括无机正磷酸盐、无机焦磷酸盐和无机多磷酸盐。无机正磷酸盐在土壤无机磷组分中占主导地位,干旱会降低无机正磷酸盐含量;无机焦磷酸盐和无机多磷酸盐可存在于高施磷水平(P₄)的土壤中。有机磷组分中,正磷酸单酯占主导地位,干旱影响紫花苜蓿对土壤中正磷酸单酯的转化和利用。综上,合理的水肥管理可对蒙东地区紫花苜蓿土壤磷营养的转化和利用进行有效的调控。     

A simple colorimetric assay method for pyrophosphate in the presence of a 1000-fold excess of orthophosphate: application of the method to the study of pyrophosphate metabolism in mitochondria

A sensitive colorimetric method for the assay of inorganic pyrophosphate with excess of orthophosphate is described. The principle of this method lies in the formation of phosphomolybdate and PPi-molybdate complexes with subsequent extraction of the phosphomolybdate complex by organic solvents and reduction of the PPi-molybdate complex by dithiothreitol and Eikonogen. The sensitivity of the method was from 5 to 120 nmol of PPi in a 2.0-ml sample.     

Crystalline inorganic pyrophosphatase isolated from baker's yeast

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 degrees C. and pH 7.0 at a concentration of 3 to 4 x 10(-3)M Na(4)P(2)O(7) and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na(4)P(2)O(7) or K(4)P(2)O(7) 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.     

On an Early Gene for Membrane-Integral Inorganic Pyrophosphatase in the Genome of an Apparently Pre-LUCA Extremophile,the Archaeon Candidatus Korarchaeum cryptofilum

A gene for membrane-integral inorganic pyrophosphatase (miPPase) was found in the composite genome of the extremophile archaeon Candidatus Korarchaeum cryptofilum (CKc). This korarchaeal genome shows unusual partial similarity to both major archaeal phyla Crenarchaeota and Euryarchaeota. Thus this Korarchaeote might have retained features that represent an ancestral archaeal form, existing before the occurrence of the evolutionary bifurcation into Crenarchaeota and Euryarchaeota. In addition, CKc lacks five genes that are common to early genomes at the LUCA border. These two properties independently suggest a pre-LUCA evolutionary position of this extremophile. Our finding of the miPPase gene in the CKc genome points to a role for the enzyme in the energy conversion of this very early archaeon. The structural features of its miPPase indicate that it can pump protons through membranes. An miPPase from the extremophile bacterium Caldicellulosiruptor saccharolyticus also has a sequence indicating a proton pump. Recent analysis of the three-dimensional structure of the miPPase from Vigna radiata has resulted in the recognition of a strongly acidic substrate (orthophosphate: Pi, pyrophosphate: PPi) binding pocket, containing 11 Asp and one Glu residues. Asp (aspartic acid) is an evolutionarily very early proteinaceous amino acid as compared to the later appearing Glu (glutamic acid). All the Asp residues are conserved in the miPPase of CKc, V. radiata and other miPPases. The high proportion of Asp, as compared to Glu, seems to strengthen our argument that biological energy conversion with binding and activities of orthophosphate (Pi) and energy-rich pyrophosphate (PPi) in connection with the origin and early evolution of life may have started with similar or even more primitive acidic peptide funnels and/or pockets.     

RIBONUCLEIC ACID-POLYPHOSPHATE FROM ALGAE: III. HYDROLYSIS STUDIES

RNA-polyphosphate was isolated from synchronous Chlorella cells.After each of a series of hydrolytic treatments, RNA-polyphosphatewas chromatographically analyzed by means of a two-column ion-exchangesystem. Alkaline hydrolysates contained primarily ribonu-cleotides,pyrophosphate, and tripolyphosphate. Acid hydrolysates containedribonucleotides, purine bases, ribonucleosides, orthophosphate,and an unknown, inorganic, phosphorus-containing compound (X-P).Treatments with pancreatic ribonuclease, spleen phosphodiesterase,and yeast polyphosphatase left large amounts of RNA-polyphosphatefragments. Treatment with venom phosphodiesterase yielded ahigh molecular weight inorganic polyphosphate fraction freefrom RNA. Such material was hydrolyzed to pyro- and tripolyphosphateby potassium hydroxide, to orthophosphate and an unknown compoundX-P by perchloric acid, and to ortho-, pyro-, and tripolyphosphateby hydroxylamine under ester hydrolysis conditions. Syntheticinorganic polyphosphate was stable to potassium hydroxide andhydroxylamine under the same conditions and yielded only orthophosphateupon perchloric acid hydrolysis. Both natural and syntheticpolyphosphates were hydrolyzed to low molecular weight fragmentsby yeast polyphosphatase. The evidence at present indicatesthat in Chlorella polyphosphate is not a simple phosphate anhydridechain. (Received June 14, 1965; )     

The biochemical conversions of conjugated dienoic and trienoic fatty acids

The changes in phosphate metabolism induced in yeast by transition from fermentation to respiration have been studied. Orthophosphate added to respiring or fermenting yeast suspensions as Na₂HP³²O₄ is rapidly resorbed and incorporated into adenosine triphosphate (ATP) and other acid-labile fractions. During fermentation, the specific activity of the orthophosphate is higher than that of ATP. This is thought to be mainly due to a heterogeneity in the intracellular orthophosphate. In respiring yeast, pyrophosphate is formed. The specific activity of this pyrophosphate is very high when the cells are maintained from the start of the experiment under aerobic conditions. When respiration follows a prior period of fermentation lasting 30–60 min., an accumulation of lowly labeled pyrophosphate occurs. Concurrently an acidinsoluble phosphate fraction is mobilized. As indicated by labeling relations, this fraction may be an intermediary in the pathway between orthophosphate and pyrophosphate. The possible role of dinucleotides in primary aerobic phosphorylation is reviewed and it is shown that diphosphopyridine nucleotide (DPN) undergoes a temporary resynthesis in yeast during the first 5–6 hr. of respiration. The question whether this phenomenon may be regarded as a secondary consequence of an enzymatic adaptation which involves pyrophosphate accumulation is discussed.     

Identification of histidine-119 as the target in the site-specific inactivation of ribonuclease A by ferrate ion.

Ferrate ion, a powerful oxidant which is an analog of orthophosphate ion, has previously shown some promise as a site-specific probe of enzymes which interact with phosphate compounds. In order to explore the general applicability of this reagent, it has been tested against ribonuclease A, an enzyme whose structure and active center have been well described. Treatment with a molar ratio of ferrate to enzyme of less than 20 leads to a loss of 87% of the activity. The known competitive inhibitors, 2'-cytidylic acid, inorganic pyrophosphate, and orthophosphate all protect the enzyme from inactivation. Inactivation is accompanied by a loss of the capacity to bind 2'-cytidylic acid. Ferrate inactivation at pH 5.0 is accompanied by the modification of only one amino acid. The amino acid which was identified by amino acid and sequence analyses of peptide fragments obtained by cyanogen bromide treatment and selective proteolysis proved to be histidine-119, whose essential role at the active center has long been established.     

Pyrophosphate-stimulated uptake of calcium into the germlings of Phytophthora infestans.

The uptake of 1 micrometer calcium into 6-h-old germination tubes of the fungus Phytophthora infestans follows Michaelis-Menten kinetics with a Km of 33 +/- 4 micrometer and a V of 0.3 nmol.min-1.(5 x 10(4) cells)-1.Uptake is inhibited by ruthenium red and lanthanum (both at 1 micrometer) and by the proton conductors 2,4-dinitrophenol (1 mM) and carbonylcyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenylhydrazone (1--10 micrometer) and also by sodium azide. These data suggest that calcium uptake is dependent on energy and on a carrier. Calcium uptake is stimulated by pyrophosphate but not by ATP, orthophosphate, or polyphosphate. This stimulation is prevented by proton conductors or by incubation at 0 degrees C.