Inorganic Pyrophosphatase Activity Research Articles

PPi Formation by Reversal of the Tonoplast-bound H+-pyrophosphatase from `Valencia' Orange Juice Cells

Tonoplast vesicles isolated from juice cells of mature `Valencia' oranges [Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (V-PPiase) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ΔpH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PPiase was not able to significantly amplify the existing ΔpH. Although not able to function as a H+ pump, V-PPiase effectively synthesized PPi in the presence of inorganic phosphate (Pi). Formation of PPi by V-PPiase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PPiase from mung bean [Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PPiase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PPiase of mature orange juice cells acts as a source of inorganic pyrophosphate (PPi) but not as a H+ pump. We propose that synthesis of PPi provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage.

Inorganic pyrophosphatase is a component of the Drosophila nucleosome remodeling factor complex.

The Drosophila nucleosome remodeling factor (NURF) is a protein complex consisting of four polypeptides that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The 140-kD NURF subunit, imitation switch (ISWI), is related to the SWI2/SNF2 ATPase. Another subunit, NURF-55, is a 55-kD WD repeat protein homologous to the human retinoblastoma-associated protein RbAp48. Here, we report the cloning and characterization of the smallest (38 kD) component of NURF. NURF-38 is strikingly homologous to known inorganic pyrophosphatases. Both recombinant NURF-38 alone and the purified NURF complex are shown to have inorganic pyrophosphatase activity. Inhibition of the pyrophosphatase activity of NURF with sodium fluoride has no significant effect on chromatin remodeling, indicating that these two activities may be biochemically uncoupled. Our results suggest that NURF-38 may serve a structural or regulatory role in the complex. Alternatively, because accumulation of unhydrolyzed pyrophosphate during nucleotide incorporation inhibits polymerization, NURF may also have been adapted to deliver pyrophosphatase to chromatin to assist in replication or transcription by efficient removal of the inhibitory metabolite.

Inhibition of Inorganic Pyrophosphatase by Phosphonate—A Site of Action inPhytophthoraspp.?

The sensitivity of inorganic pyrophosphatase (EC 3.6.1.1) to phosphonate was compared in microorganisms, plants, and mammals. Phosphonate inhibited inorganic pyrophosphatase activity regardless of source, with IC50values ranging from 39 (human cell lines) to 565 mM (rat liver) when assayed at saturating substrate concentration. The IC50values for phosphonate inhibition of the pyrophosphatases fromPhytophthora palmivoraandPhytophthora melonis, plant pathogens susceptible to phosphonate in the field, were 109 and 131 mM, respectively. At a substrate concentration close to theKmvalue (40 μM), phosphonate gave an IC50of 34 ± 15 mM against the pyrophosphatase fromP. palmivora, slightly lower than the corresponding IC50value for the pyrophosphatase fromSaccharomyces cerevisae, 85 ± 7 mM. Phosphate, the product of pyrophosphatase and a known allosteric inhibitor of the enzyme inS. cerevisae, proved to be a slightly better inhibitor of theP. palmivorapyrophosphatase than phosphonate (IC50of 15 ± 3 mM). Sensitivity to both phosphonate and phosphate was dependent upon the concentration of Mg2+, and increasing concentrations of Mg2+reduced, but never completely abolished, inhibition. Our results suggest that at the levels known to accumulate withinPhytophthoramycelia, phosphonate could inhibit pyrophosphatase activityin vivo. However, since the inorganic pyrophosphatases from yeast andPhytophthorawere almost equally sensitive to phosphonatein vitro, whilePhytophthoraspp. are much more sensitive to phosphonatein vivothan yeast, we conclude that this enzyme is unlikely to be the site at which phosphonate provides specificity againstPhytophthoraspp. when compared to other microorganisms.

Guanylyltransferase and RNA 5′-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus

We have examined the RNA-capping enzyme activities of bluetongue virus (BTV) minor core protein, VP4. Recombinant BTV VP4 protein was purified to homogeneity from insect cell culture infected with a baculovirus VP4 of BTV serotype 10. We demonstrate that the purified protein, and VP4 encapsidated in core-like particles, react with GTP and covalently bind GMP via a phosphoamide linkage, a characteristic feature of guanylyltransferase enzyme. VP4 also catalyses a GTP-PPi exchange reaction indicating that the protein is the guanylyltransferase of the virus. In addition, VP4 possesses an RNA 5′-triphosphatase activity which catalyses the first step in the RNA-capping sequence. Further, an inorganic pyrophosphatase activity was identified which may aid the transcription activity within the virus by removing inorganic pyrophosphate which is an inhibitor of the polymerization reaction. Finally, the direct evidence of VP4 capping activity has been obtained by demonstrating in vitro transfer of GMP to the 5′ end of in vitro synthesized BTV ssRNA transcripts to form a cap structure.

Cadmium Suppresses Phosphate Level and Inhibits the Activity of Phosphatases in Growing Rice Seedlings

AbstractThe effect of increasing concentration of Cd in situ on the metabolic status of total phosphate and the activity of its metabolizing enzymes acid phosphatase, alkaline phosphatase and inorganic pyrophosphatases were examined in growing rice (Oryza sativa L.) seedlings. Some 500 μm Cd in the medium caused 68–77% decline in phosphate content in shoots and 56–66% decline in roots in 20‐day‐old seedlings of the two rice cultivars Ratna and Jaya. In situ Cd levels of 100 μm and 500 μm led to a significant decline in the activities of the three phosphorolytic enzymes studied. Inhibition in the acid phosphatase activity was greater in roots than in shoots. With 500 μm Cd 62–88% inhibition in acid phosphatase activity was observed in roots. A similar Cd level caused 28–31% inhibition in shoots in 20‐day‐old seedlings, whereas inorganic pyrophosphatase activity was inhibited by 27–53% in roots and nearly 50% in shoots. Under in vitro conditions more than 200 μm Cd(NO3)2 in the reaction medium significantly inhibited the activities of the phosphorolytic enzymes. Alkaline phosphatase appeared to be more tolerant than acid phosphatase at lower (20–100 μm) concentrations whereas the activity of inorganic pyrophosphatase was completely lost with 2 mM Cd. Isoenzymic studies revealed three acid phosphatase isoenzymes with RF values of 0.18, 0.24 and 0.40 in both roots and shoots. The band intensities decreased under Cd treatments. The results indicate suppression of the activities of phosphatases due to Cd in growing seedlings.

Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3.

Each of two histidine residues and one tryptophan residue in thermophilic bacterium PS-3 inorganic pyrophosphatase (PPase) was replaced by alanine. The activities of the H125A and W143A variants decreased to one-fifth, whereas the activity of H118A remained unaltered. CD spectra in the near-UV region indicated that the conformations of the first two variants changed with the substitution. In contrast with wild-type PPase, which is hexameric beyond an enzyme concentration of 0.1 microM in the presence of Mg2+, the H118A and H125A variants cannot be assembled from trimers into hexamers at less than an enzyme concentration of 10 microM even at a higher concentration of Mg2+. In particular, H118A was irreversibly inactivated in a diluted state. In contrast, the enzyme concentration dependence of W143A PPase activity was almost the same as that of wild-type PPase. These results indicated that His-118 and His-125 are important for both trimer-trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer-trimer interaction is absolutely necessary for the thermostability of the PS-3 enzyme.

Chlorogenic Acid Analogue S 3483: A Potent Competitive Inhibitor of the Hepatic and Renal Glucose-6-Phosphatase Systems

S 3483, a synthetic derivative of chlorogenic acid (CHL), was found to be a reversible, linear competitive inhibitor of the glucose-6-phosphatase (Glc-6-Pase) system in rat renal microsomes and rat and human liver microsomes. TheKifor S 3483 in rat liver microsomes (129 nM) is three orders of magnitude smaller than theKifor CHL. S 3483 up to 100 μM had no effect on the Glc-6-Pase enzyme activity or on the system inorganic pyrophosphatase activity (i.e., on T2, the Pi/inorganic pyrophosphate transporter). Thus, like CHL, S 3483 appears to be a site-specific inhibitor of T1, the Glc-6-P transporter of renal and liver microsomes. The potency of S 3483 was unaffected when the ratioVmax(T1):Vmax(enzyme)was altered over a 10-fold range by applying enzyme inhibition and selective inactivation of T1. The absence of T1-imposed rate restrictions on the potency of reversible T1 inhibitors contrasts markedly with the response of reversible Glc-6-Pase enzyme inhibitors, whose potency declines sharply as T1 becomes more rate controlling. The potency of S 3483, but not of CHL, decreased as the microsomal protein concentration in the assay medium was increased. This effect suggests that as the protein concentration was raised the concentration of T1 in the assay medium approached the order of magnitude of theKifor S 3483. Thus, the microsomal content of T1 is likely to be on the order of 100 pmol/mg protein. S 3483 is the most potent inhibitor of the Glc-6-Pase system reported to date. It and other tight-binding inhibitors of T1 will provide useful new tools for investigating the molecular structure and physiology/pathology of the Glc-6-Pase system.

A New Enzymatic Method for Assaying Serum Inorganic Pyrophosphatase.

We developed a novel simple method to determine serum inorganic pyrophosphatase (PPase; EC 3.6.1.1) activity using purine nucleoside phosphorylase, xanthine oxidase and peroxidase, which method is applicable to automated analysis. Good reproducibility was demonstrated, with a coefficient of variation of 2.5 to 4.0% depending on the PPase activity. Good stability after color development was also observed. The presence of ascorbic acid, bilirubin, hemoglobin or intra-lipid did not influence the results. We also investigated the relationship between the activities of alkaline phosphatase (ALP; EC 3.1.3.1) and PPase. A strong positive correlation between ALP from bone and placenta and PPase was demonstrated with a correlation coefficient of 0.932 and 0.909, respectively. In contrast, ALP from liver showed a relatively weak correlation, with a correlation coefficient of 0.839. Further, placenta showed a higher ratio of PPase activity to ALP activity than did other tissues. PPase and ALP could not be distinguished by gel filtration or by ion-exchange chromatography.

Cadmium Alters Phosphate Level and Suppresses Activity of Phosphorolytic Enzymes in Germinating Rice Seeds

AbstractThe effect of increasing concentration of Cd on the metabolic status of phosphate and the activity of its metabolizing enzymes acid phosphatase, alkaline phosphatase and inorganic pyrophosphatase were examined in germinating rice (Oryza sativa) seeds. During a 0‐120 h germination period in the two rice cvs., Ratna and Jaya, 500 μM Cd caused a 2‐3‐times increase in phosphate level in embryo axes compared to controls. Cadmium concentration of 500 μM in situ caused about 12‐14% and 20‐30 % decline in acid phosphatase activity in endosperms and embryo axes respectively during 72‐96 h of germination. With 500 μM Cd treatment in situ about 27‐43 % inhibition in alkaline phosphatase activity was noticed in endosperms and 40‐70 % in embryo axes. With 2 mM Cd salt in vitro about 60‐92 % inhibition in the activity of acid phosphatase and 80‐95 % inhibition of alkaline phosphatases was observed in enzyme preparations from embryo axes. However, a Cd level of 50‐100 μM caused stimulation in the activity of inorganic pyrophosphatase. Isoenzymic studies revealed four acid phosphatase isoenzymes in embryo axes with Rf values 0.15, 0.24, 0.36 and 0.49 and three isoenzymes in endosperms with Rf values 0.18, 0.24 and 0.40. The band intensities decreased under Cd treatment. Results indicate possible suppression of the phosphorolytic activities in germinating rice seeds under Cd stress.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2.

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an alpha2beta2gamma2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels ( approximately 10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 micromol acetoacetate formed min-1.mg-1 at 30 degrees C and pH 7.6 and apparent Km values of 7.80 microM (acetone), 122 microM (ATP), and 4. 17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

Chlorogenic Acid and Hydroxynitrobenzaldehyde: New Inhibitors of Hepatic Glucose 6-Phosphatase

We have studied the interactions of chlorogenic acid (CHL) and 2-hydroxy-5-nitrobenzaldehyde (HNB) with the components of the rat hepatic glucose 6-phosphatase (Glc-6-Pase) system. CHL and HNB are competitive inhibitors of glucose 6-phosphate (Glc-6-P) hydrolysis in intact microsomes withKivalues of 0.26 and 0.22 mm, respectively. CHL is without effect on the enzyme of fully disrupted microsomes or the system inorganic pyrophosphatase (PPiase) activity. HNB is a potent competitive inhibitor of the system PPiase activity (Ki= 0.56 mm) and a somewhat weaker noncompetitive inhibitor of enzyme activity (Ki= 2.1 mm). These findings indicate CHL binds to T1, the Glc-6-P transporter, and HNB inhibits through interaction with both T1 and T2 the phosphate (Pi)–PPitransporter. Binding of CHL and HNB is freely reversible. However, the inhibition of both PPiase and Glc-6-Pase by HNB becomes irreversible following incubation of HNB-exposed microsomes with 2.5 mmsodium borohydride, indicating that inhibition involves the formation of a Schiff base. The presence of CHL effectively protects T1, but not T2, against the irreversible inhibition by HNB. In contrast, PPiand Piare effective in protecting T2, but not T1. This is the first report describing an effective inhibitor of the system PPiase activity (T2). CHL is the most specific T1 inhibitor described to date.

Effect of tillage and residue management on enzyme activities in soils: III. Phosphatases and arylsulfatase

This study was carried out to investigate the effect of tillage and residue management on activities of phosphatases (acid phosphatase, alkaline phosphatase, phosphodiesterase, and inorganic pyrophosphatase) and arylsulfatase. The land treatments included three tillage systems (no-till, chisel plow, and moldboard plow) in combination with corn residue placements in four replications. The activities of these enzymes in no-till/double mulch were significantly greater than those in the other treatments studied, including no-till/bare, no-till/normal, chisel/normal, chisel/mulch, moldboard/normal, and moldboard/mulch. The effect of mulching on activities of phosphatases was not as significant as on activities of arylsulfatase. The lowest enzyme activities were found in soil samples form no-till/bare and moldboard/normal treatments, with the exception of inorganic pyrophosphatase, which showed the lowest activity in no-till/bare only. Among the same residue placements, no-till and chisel plow showed comparable arylsulfatase activity, whereas the use of moldboard plow resulted in much lower arylsulfatase activity. The activities of phosphatases and arylsulfatase were significantly correlated with organic C in the 40 soil samples studies, with r values ranging from 0.71*** to 0.92***. The activities of alkaline phosphatase, phosphodiesterase, and arylsulfatase were significantly correlated with soil pH, with r values of 0.85***, 0.78***, and 0.77***, respectively, in the 28 surface soil samples studied, but acid phosphatase and inorganic pyrophosphatase activities were not significantly correlated with soil pH. The activities of phosphatases and arylsulfatase decreased markedly with increasing soil depth and this decrease was associated with a decrease in organic C content. The activities of these enzymes were also significantly intercorrelated, with r values ranging from 0.50*** to 0.92***.

Effect ofCrataeva nurvala bark decoction on enzymatic changes in liver of normal and stone forming rats

The influence of Crataeva nurvala bark decoction was studied in calcium oxalate stone forming rats, in relation to oxalate metabolism in liver. The activities of the major oxalate synthesizing enzymes in liver namely, glycollate oxidase (GAO) and lactate dehydrogenase (LDH) were significantly increased in the calculogenic group. Bark decoction treatment lowered the liver GAO activity considerably. Transport ATPases (Na+, K+ and Ca2+-ATPases) and alkaline phosphatase were enhanced in rats fed calculi producing diet, while the activities of acid phosphatase, inorganic pyrophosphatase and aminotransferases were slightly reduced. Bark decoction administration produced a marginal decrease in Na+, K+-ATPase and increase in aspartate aminotransferase activities, without significantly altering other enzyme activities. The decrease in liver GAO activity seen during bark decoction treatment, with concomitant decrease in kidney oxalate level, may prove beneficial as a prophylactic measure in preventing stone recurrence.

Characterization and partial purification of an ATPase and inorganic pyrophosphate of the archaebacterium Methanobacterium thermoautotrophicum

ATPase and inorganic pyrophosphatase (PPase) activities have been detected in several methanogenic bacteria. These activities are believed to play a crucial role in energy metabolism. In the present study we have investigated some characteristics of the ATPase and the inorganic PPase activities of Methanobacterium thermoautotrophicum. Although these proteins migrate identically on non-dissociating gels, they are catalyzed by distinct enzymes which are separable by biochemical purification methods. The partially purified enzymes are composed of at least two subunits. The ATPase subunits have molecular masses of about 43 and 33 kDa and the inorganic PPase such of about 31 and 25 kDa. After purification, the PPase and the ATPase did not hydrolyze ATP, and PP i, respectively. The membrane-bound ATPase and PPase activities are distinguished in response to sodium fluoride, by the effects of divalent cations, by the temperature ranges for activities and the solubilization behaviour by different extractants. Most investigated catalytic and structural properties of the ATPase do not suit the current criteria for classifying this enzyme under the F-, V- or P-ATPases.

Time-dependent inhibition of glucose 6-phosphatase by 3-mercaptopicolinic acid

3-Mercaptopicolinate (3-MP) inhibits d-glucose-6-phosphate (G6P) phosphohydrolase activity of the glucose-6-phosphatase system (Bode et al. (1993) Biochem. Cell Biol. 71, 113–121). We therefore attempted to maximize the inhibition by varying the physical state of microsomes, the concentration of 3-MP, and the time of preliminary incubation of 3-MP with the enzyme. The inhibition was irreversible and time- and inhibitor-concentration-dependent, with G6P phosphohydrolase activity of intact rat liver microsomes, but there was no inhibition with detergent-treated microsomes. The effectiveness of 3-MP as a time-dependent inhibitor of glucose 6-phosphatase was demonstrated in situ by measuring glycogenolysis in isolated, perfused livers from fed rats. We first exposed the livers to 2 mM 3-MP for 40 min, and then assessed the inhibitory effects on glycogenolysis. It was lowered by 50%. These observations establish that 3-MP at the mM level may be useful as an experimental probe in the study of the role(s) of G6P in the regulation of glycogenolysis as well as glycogenesis. Further, they validate the use of much lower (μM) concentrations of 3-MP to block gluconeogenesis (at the phospho enolpyruvate carboxykinase step) without interfering with glucose 6-phosphatase. We also explored the mechanism of 3-MP inhibition. The time-dependent inhibition of carbamoyl-phosphate:glucose phosphotransferase activity with microsomes incubated with 1 mM 3-MP for 60 or 90 min and then assayed with 1 mM carbamoyl phosphate and 180 mM glucose was modest compared with inhibition of G6P phosphohydrolase. When G6P production by carbamoyl-phosphate:glucose phosphotransferase was reduced by decreasing glucose concentration to 60 mM, no inhibition by 3-MP was discernible. There was no inhibition of inorganic pyrophosphatase activity. These studies support the model of time-dependent, irreversible reaction of 3-MP with the G6P translocase component of the glucose-6-phosphatase system.

Phosphomonoesterase, phosphodiesterase, phosphotriesterase, and inorganic pyrophosphatase activities in forest soils in an alpine area: effect of pH on enzyme activity and extractability

We investigated different types of phosphatase activity (phosphomono-, phosphodi-, phosphotriesterase, inorganic pyrophosphatase) in five forest soils in Vorarlberg, Austria. Phosphatase activity was determined both in soils and in soil extracts prepared with different solutions (distilled water, 0.1M sodium pyrophosphate at pH 7, 0.1M sodium phosphate buffer/1M KCl at pH 6.5, and a modified universal buffer at pH 4, 6.5, 9, and 11). High phosphomonoesterase activity in these soils indicated a severe deficiency in available P. Acidic phosphomonoesterase prevailed over alkaline phosphomonoesterase activity. Phosphodiesterase was highest in the least acidic soil but no general trend towards an optimum pH was recognized. Phosphotriesterase activity was observed in only two of the five soils and favoured an alkaline optimum pH; this activity was not detected in strongly acid soils. Inorganic pyrophosphatase activity was high in soils with no phosphotriesterase. Phosphomonoesterase, phosphodiesterase and inorganic pyrophosphatase activities were much lower in soil extracts than in soils.

Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae.

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.

Inorganic pyrophosphatase of clofibrate-induced rat liver peroxisomes.

Clofibrate increased inorganic pyrophosphatase (PPase) activity in peroxisomes more than 12-fold (740 milliunits/head, 15.9 +/- 5.0 milliunits/mg protein) in rat liver. The distribution of cytochrome c oxidase and that of the PPase in a Nycodenz gradient suggested that the PPase is an original peroxisomal enzyme and not a mitochondrial contaminant: This was confirmed by second Nycodenz gradient centrifugation. The optimum pH of the peroxisomal PPase was about 8.5. The activity was specific to inorganic pyrophosphate (PPi), the Km value for PPi being 34.1 +/- 3.3 microM. It was strictly dependent on Mg2+ and showed a sigmoidal dose-response for Mg2+ with an S0.5 value of 100 microM. The activity was inhibited by Ca2+, p-chloromercuriphenylsulfonic acid, Hg2+, N-ethylmaleimide, and NaF. The functions of peroxisomal PPase are discussed.

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.

Hysteretic behavior of the hepatic microsomal glucose-6-phosphatase system

Carbamyl-P:glucose and PP 2:glucose phosphotransferase, but not inorganic pyrophosphatase, activities of the hepatic microsomal glucose-6-phosphatase system demonstrate a time-dependent lag in product production with 1 mM phosphate substrate. Glucose-6-P phosphohydrolase shows a similar behavior with [glucose-6-P] ≦ 1.10 mM, but inorganic pyrophosphatase activity does not even at the 0.05 or 0.02 mM level. The hysteric behavior is abolished when the structural integrity of the microsomes is destroyed by detergent treatment. Calculations indicate that an intramicrosomal glucose-6-P concentration of between 20 and 40 μM must be achieved, whether in response to exogenously added glucose-6-P or via intramicrosomal synthesis by carbamyl-P: glucose or PP 1: glucose phosphotransferase activity, before the maximally active form of the enzyme system is achieved. It is suggested that translocase T 1, the transport component of the glucose-6-phosphatase system specific for glucose-6-P, is the target for activation by these critical intramicrosomal concentration of glucose-6-P.