Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinities for fructose diphosphate and Mg²⁺ and were similarly activated by EDTA, dithiothreitol and cystamine.Mg²⁺, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

The preparation of adenosine 5′-pyrophosphate by a non-enzymic method

1. A non-enzymic method for the preparation of adenosine 5′-diphosphate is described, in which the terminal phosphate of adenosine 5′-triphosphate is transferred to methanol in the presence of hydrochloric acid. The final purified product can be obtained in 60% yield. 2. Experiments with [¹⁴C]methanol showed that no methylation of the adenosine diphosphate occurs during the reaction. 3. Confirmation that the pyrophosphate moiety of the adenosine diphosphate produced was in the 5′-position was obtained by: (a) periodate oxidation; (b) treatment with apyrase and examination of the resulting adenylic acid isomer by paper chromatography. 4. The method appears to be generally applicable to the preparation of nucleoside 5′-diphosphates from the corresponding nucleoside 5′-triphosphates.     

Adenosine triphosphatase in isolated membranes of Staphylococcus aureus

The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5′-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 μmole per min per mg of protein under optimal conditions. The enzyme was Mg⁺⁺-dependent and K⁺- or Na⁺-stimulated. Maximal activity was observed with a molar adenosine 5′-triphosphate (ATP) to Mg⁺⁺ ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5′-diphosphate. Inorganic pyrophosphate and the 5′-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.     

Further studies on the bicarbonate stimulation of photophosphorylation in isolated chloroplasts

The bicarbonate effect in stimulating the rate of photophosphorylation by isolated spinach (Spinacia oleracea var. Virginia blight-resistant savoy) chloroplasts at a pH below the optimum has been re-examined. Its seasonal nature may be related to the hormonal status of the plants. Bicarbonate anions stimulate adenosine 5′-triphosphate synthesis if added in the final, adenosine 5′-triphosphate-forming stage of either a postillumination or an acid-base experiment. They also stimulate the membrane-bound, Mg²⁺-dependent adenosine 5′-triphosphatase of chloroplasts, and the Ca²⁺-dependent adenosine 5′-triphosphatase of detached coupling factor. These and other data point to the interaction between energized thylakoid membranes and the coupling factor as the probable site of action of bicarbonate anions when they stimulate photophosphorylation.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Diacylglycerol kinase from suspension cultured plant cells : characterization and subcellular localization

Diacylglycerol kinase (adenosine 5′-triphosphate:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5′-triphosphate and guanosine 5′-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane.     

Partial purification and properties of an acid nucleotidase from the postmicrosomal supernatant of rat spleen

1. The dephosphorylation of 3′-AMP, 3′-dAMP, 3′-CMP and 3′-dCMP was studied in the postmicrosomal supernatant of rat spleen and liver. In both organs 3′-AMP and 3′-dAMP were dephosphorylated at an appreciable rate, in both the presence and the absence of Mg²⁺. The pH optimum for this dephosphorylation was in the range 4.5–5.0. 3′-CMP and 3′-dCMP were very slowly degraded, though the activity towards 3′-dCMP increased somewhat in the presence of Mg²⁺. The optimum pH for this Mg²⁺-dependent dephosphorylation was 5.5–6.0. 2. The rate of dephosphorylation of 3′-AMP and 3′-dAMP per mg of protein was about 5 times as high in spleen as in liver. 3. The dephosphorylation of 3′-AMP could be ascribed to a single enzyme with pH optimum about 4.5. The activity towards 3′-dAMP could be resolved into one component coinciding with the 3′-dAMP-degrading enzyme, and one Mg²⁺-requiring component probably identical with the soluble deoxyinosine-activated nucleotidase. The dephosphorylation of 3′-dCMP seemed to be performed only by the latter enzyme. 4. The enzyme dephosphorylating 3′-AMP was purified 200-fold from the postmicrosomal supernatant and its physical and catalytic properties were compared with those of acid nucleotidase (EC 3.1.3.31) purified from rat liver lysosomes. The two enzymes were identical in all properties tested (substrate specificity, K_m, molecular weight, response to phosphatase inhibitors), but some of the data differed from earlier reports on the acid nucleotidase. 5. The subcellular localization of the acid nucleotidase, its relationship to the acid phosphatase(s) and its role in the breakdown of nucleic acid constituents are discussed.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : V. Modulation of the Spinach Leaf Cytosolic Fructose 1,6-Bisphosphatase Activity in Vitro by Substrate, Products, pH, Magnesium, Fructose 2,6-Bisphosphate, Adenosine Monophosphate, and Dihydroxyacetone Phosphate

How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg²⁺ is required as an activator. There is a wide pH optimum, especially at high Mg²⁺. The substrate dependence is not markedly pH dependent. High concentrations of Mg²⁺ and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg²⁺ dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg²⁺ and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg²⁺-dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg²⁺, but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S_0.5 for fructose 1,6-bisphosphate. A model is proposed to account for the interaction between these effectors.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Molecular Basis for Nucleotide Conservation at the Ends of the Dengue Virus Genome

The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol_DV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol_DV contains a specific priming site for adenosine 5′-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn²⁺ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol_DV ensures the conservation of the 5′-adenosine by strongly discriminating against viral templates containing an erroneous 3′-end nucleotide in the presence of Mg²⁺. In the presence of Mn²⁺, NS5Pol_DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.     

Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5'-Monophosphate and Determination of a Short Nucleotide Sequence at the 3' End of the RNA

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5′-monophosphate and adenosine 5′-monophosphate (AMP) when it is incubated in the presence of cytidine 5′-triphosphate (CTP), adenosine 5′-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When α ³²PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3′ end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3′ end of which is found in a fragment having a sedimentation constant close to 5s.     

P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

Isolation of uridine 5'-pyrophosphate glucuronic Acid pyrophosphorylase and its assay using p-pyrophosphate

A procedure was devised to detect and assay uridine 5′-pyrophosphate (UDP)-glucuronic acid pyrophosphorylase in plant extracts. Substrates are UDP-glucuronic acid and ³²P-pyrophosphate, and the ³²P-uridine 5′-triphosphate produced is selectively adsorbed to charcoal. The charcoal adsorption procedure is a modification of that used to determine ³²P-adenosine 5′-triphosphate produced by adenosine 5′-pyrophosphate glucose pyrophosphorylase, and the modification greatly improves the retention of uridine 5′-triphosphate.     

Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves

The activity of adenosine 5′ triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, ³⁵SO₄²⁻ incorporation into adenosine 5′ phosphosulfate, and ATP synthesis dependent upon adenosine 5′ phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO₃⁻ and CO₂ assimilation in leaves of C₄ plants.     

Models describing the kinetics of ribulose biphosphate carboxylase-oxygenase.

Equations are developed to describe the reactions of ribulose 1,5-biphosphate carboxylase—oxygenase with ribulose biphosphate (RuP₂), carbon dioxide, and oxygen. It is predicted that at the high concentrations of enzyme sites found in vivo there will be a large proportion of the total RuP₂ bound to the enzyme. The kinetic characteristics of the in vivo reactions with RuP₂ are predicted to be analogous to those which would occur in the presence of a tight-binding substrate. Equations are developed which are applicable when the enzyme is only partially activated by CO₂ and Mg²⁺. The response of carboxylase velocity to CO₂ concentration is sigmoidal when Mg²⁺ concentration is low.     

Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S_0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K_m value. The enzyme showed an absolute requirement for divalent cations (either Mg²⁺ or Mn²⁺), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg²⁺-activated enzyme to varying degrees (Ni²⁺ > Zn²⁺ > Cu²⁺ > Ca²⁺ > Ba²⁺). The differences in the kinetic responses of the enzyme to Mg²⁺ and Mn²⁺ are discussed.     

Activation of rabbit kidney fructose diphosphatase by Mg-EDTA, Mn-EDTA and Co-EDTA complexes

Purified rabbit kidney fructose diphosphatase requires both a free cation and a metal-chelate when assayed at pH 8 or below. In the presence Mg²⁺ or Mn²⁺, effective metal chelates were Mn(II)-EDTA, Mg(II)-EDTA, and Co(III)-EDTA. With Mg²⁺ as the cation the affinity of the enzyme for Mn(II)-EDTA or Mg(II)-EDTA was approximately the same, and 300-fold greater than that for Co(III)-EDTA.Activation of the enzyme by the very stable Co(III)-EDTA complex, as well as failure of an ionophore antibiotic to replace EDTA as activator, exclude the possibility that the effects of EDTA are due to removal of metal inhibitors.Inhibition of fructose diphosphatase by Ca²⁺ was competitive with Mg²⁺, and noncompetitive with Mg(II)-EDTA, or Co(III)-EDTA. Conversely inhibition by Zn(II)-EDTA was competitive with Mg(II)-EDTA and noncompetitive with free Mg²⁺. The data suggest that the free metals bind to one site on the enzyme while the metal-EDTA chelates bind to a second site.     

Hymenolepis diminuta: Partial characterization of the membrane-bound and solubilized alkaline phosphohydrolase activities of the isolated brush border plasma membrane

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate (p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg²⁺ (1 mM), although low concentrations of Mn²⁺, Ca²⁺, or Zn²⁺ will also partially satisfy this requirement; higher concentrations of Mg²⁺ and Mn²⁺, and other divalent cations (Cu²⁺, Fe²⁺, and Pb²⁺), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and K_m, but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F^?, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.