Properties of cotton inorganic pyrophosphatase

The activity of inorganic pyrophosphatase and pyrophosphate content were studied in developing and germinating cotton seeds. It was shown that the content of pyrophosphate in germinating seeds reached its maximum value after two days of their development, and the activity of inorganic pyrophosphatase, one day after the beginning of seed bud formation. The low pyrophosphatase activity of dormant seeds increased during their germination under open-ground conditions, reaching its maximum on day 6-7. Properties of partly purified pyrophosphatase from three-day-old cotton seedlings grown under laboratory conditions were studied.     

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.     

Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.     

The Effect of Pyrophosphate Analogues on the Activity of the Inorganic Pyrophosphatase from Escherichia coli

The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E. coli was studied. Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate.     

The effect of pyrophosphate analogues on the inorganic pyrophosphatase from Escherichia coli

The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E. coli was studied. Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.     

Some characteristics of cotton pyrophosphatase activation by magnesium

The effect of Mg²⁺ ions on inducing pyrophosphatase activity of germinating cotton (Gossypium hirsutum L.) seeds was investigated. The presence of Mg²⁺ ions in the germination medium markedly shortened time for the attainment of the pyrophosphatase maximum activity (T _max). In the absence of Mg²⁺ ions in the nutrient medium, T _max comprised 6.0–6.5 days, whereas in the presence of 3–5 mM Mg²⁺, T _max was decreased to 3–4 days. An increase in the concentration of Mg²⁺ ions in the medium up to 5 mM resulted in an increase in pyrophosphatase activity. The effect of Mg²⁺ ions on the activity of a purified pyrophosphatase preparation isolated from three-day-old cotton seedlings was investigated. Mg²⁺ ions did not affect the rate of attainment of a maximum pyrophosphatase activity, but decreased the value of the Michaelis-Menten constant.     

Sucrose metabolism in lima bean seeds

Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.     

Na2CO3胁迫下青海星星草种子萌发过程中淀粉酶活性等的变化

对Na2CO3胁迫下青海星星草种子萌发过程中淀粉酶活性、可溶性糖含量、呼吸强度的变化进行了研究。结果表明在Na2CO3胁迫下,种子萌发过程中淀粉酶活性、可溶性糖含量、呼吸强度与空白对照相比均下降,当Na2CO3浓度大于5.00%时,青海星星草种子在播种后至测量结束时,淀粉酶活性、可溶性糖含量、呼吸强度几乎都没有变化,三者与Na2CO3胁迫浓度的负相关关系均极其显著。在播种后的1～8d可溶性糖含量的变化与淀粉酶活性的变化基本趋于一致,两者呈显著的正相关关系。第8天以后,可溶性糖含量开始降低,而淀粉酶活性继续升高,说明可溶性糖已被利用、转化或合成新物质,Na2CO3胁迫下可溶性糖含量减少主要是因为淀粉酶活性受到了抑制。水解酶活性降低、储藏物质不能动员导致其不能水解、呼吸代谢受抑制是Na2CO3胁迫下青海星星草种子萌发受抑制的主要原因。     

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.     

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.     

Alkaline inorganic pyrophosphatase and starch synthesis in amyloplasts

The aim of this work was to see if amyloplasts contained inorganic pyrophosphatase. Alkaline pyrophosphatase activity, largely dependant upon MgCl₂ but not affected by 100 M ammonium molybdate or 60–100 mM KCl, was demonstrated in exracts of developing and mature clubs of the spadix of Arum maculatum L. and of suspension cultures of Glycine max L., but not in extracts of the developing bulb of Allium cepa L. The maximum catalytic activity of alkaline pyrophosphatase in the above tissues showed a positive correlation with starch synthesis, and in the first two tissues was shown to exceed the activity of ADPglucose pyrophosphorylase. Of the alkaline pyrophosphatase activity in lysates of protoplasts of suspension cultures of Glycine max, 57% was latent. Density-gradient centrifugation of these lysates showed a close correlation between the distribution of alkaline pyrophosphatase and the plastid marker, nitrite reductase. It is suggested that much, if not all, of the alkaline pyrophosphatase in suspension cultures of Glycine max is located in the plastids.Abbreviations PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean

Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAE-Toyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+ and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited by ATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+ concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N'-Dicyclohexylcarbodiimide became bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.     

Zn2+-Dependent Acid Inorganic Pyrophosphatase Activity as Related to Other Pyrophosphatases in Oryza sativa

Acid inorganic pyrophosphatase on the one hand, and Mg²⁺-dependent alkaline inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase on the other hand showed opposite trends in their activities in rice (Oryza sativa L. cv. Ratna) seedlings grown in dark and sun. The opposite trends in their activities were also noted in rice seedlings grown from gamma-irradiated seeds and in detached rice leaves floated on water in dark. The ratios of Mg²⁺ dependent alkaline inorganic pyrophosphatase/acid inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase/acid inorganic pyrophosphatase changed significantly in response to the above physical treatments, but the ratio of Mg²⁺ dependent alkaline inorganic pyrophosphatase/Zn²⁺ dependent acid inorganic pyrophosphatase remained relatively stable. The conclusion is that Zn²⁺-dependent acid inorganic pyrophosphatase activity is the same as that of Mg²⁺-dependent alkaline inorganic pyrophosphatase and is different from that of acid inorganic pyrophosphatase, which requires no metal ion for activity. The acid and alkaline inorganic pyrophosphatase activities are due to separate enzyme proteins.     

Metabolism of trypsin-inhibitory proteins in the germinating seeds of kidney bean (Phaseolus vulgaris)

Summary A number of proteins with trypsin-inhibitory activity was separated by isoelectric focusing and their amounts measured in the extracts of the seeds of kidney bean at various stages of germination up to 16 days.The total trypsin inhibitor content of the dormant seed, 2.2 mg per g bean rose to about 3.6 mg by the seventh day and declined slowly after the tenth day of germination. The individual trypsin inhibitors however, appeared to change independently of each other and some components disappeared almost completely with the progress of germination. The emergence of an inhibitor not found in the dormant seed was also observed. Some of the inhibitor proteins attained a maximum concentration by the 7–8th day of germination. This coincided with a similar maximum in the general protein and proteolytic enzyme content of the germinating bean seeds. The results obtained suggested that the main function during germination of these protein components might not be related to their trypsin-inhibitory activity.     

Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.     

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Acid phosphatase activities during the germination of Glycine max seeds.

In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.     

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.     

On the Germination and Changes in Chemical Composition During Seedling Emergence and Development of Cotton Seeds

Seeds from two cotton varieties attained similar maximum germinationpercentages at 30°C. Failure of germination occurred at15 and 45°C. At lower and higher temperatures than 30°Cmaximum germination percentages were lower and the periods beforegermination was intiated longer. Fumigation of seeds with methylbromide decreased their maximum emergence percentages in thefield, but dressing seeds with mercuric compounds had no effecton emergence. During the first 20 days of seed development theaccumulation of total dry-matter and oil was rapid and thenit continued at a steady and slower rate till day 52. A linearrelationship existed between the oil and residual dry-mattercontent of developing seeds. The disappearance of oil from thecotyledons of germinating seeds was gradual over the periodof 5 days of germination while that of starch was very rapidduring the first day, thereafter very little starch was mobilizedfrom the cotyledons to the embryo.