Demonstration of 220/280 GHz Multichroic Feedhorn-Coupled TES Polarimeter.

We describe the design and measurement of feedhorn-coupled, transition-edge sensor (TES) polarimeters with two passbands centered at 220 GHz and 280 GHz, intended for observations of the cosmic microwave background. Each pixel couples polarized light in two linear polarizations by use of a planar orthomode transducer and senses power via four TES bolometers, one for each band in each linear polarization. Previous designs of this detector architecture incorporated passbands from 27 to 220 GHz; we now demonstrate this technology at frequencies up to 315 GHz. Observational passbands are defined with an on-chip diplexer, and Fourier-transform-spectrometer measurements are in excellent agreement with simulations. We find coupling from feedhorn to TES bolometer using a cryogenic, temperature-controlled thermal source. We determine the optical efficiency of our device is η = 77% ± 6% (75% ± 5%) for 220 (280) GHz, relative to the designed passband shapes. Lastly, we compare two power-termination schemes commonly used in wide-bandwidth millimeter-wave polarimeters and find equal performance in terms of optical efficiency and passband shape.

Optimization of Time- and Code-Division-Multiplexed Readout for Athena X-IFU.

Readout of a large, spacecraft-based array of superconducting transition-edge sensors (TESs) requires careful management of the layout area and power dissipation of the cryogenic-circuit components. We present three optimizations of our time- (TDM) and code-division-multiplexing (CDM) systems for the X-ray Integral Field Unit (X-IFU), a several-thousand-pixel-TES array for the planned Athena-satellite mission. The first optimization is a new readout scheme that is a hybrid of CDM and TDM. This C/TDM architecture balances CDM's noise advantage with TDM's layout compactness. The second is a redesign of a component: the shunt resistor that provides a dc-voltage bias to the TESs. A new layout and a thicker Pd-Au resistive layer combine to reduce this resistor's area by more than a factor of 5. Third, we have studied the power dissipated by the first-stage SQUIDs (superconducting quantum-interference devices) and the readout noise versus the critical current of the first-stage SqUIDs. As a result, the X-IFU TDM and C/TDM SQUIDs will have a specified junction critical current of 5 µA. Based on these design optimizations and TDM experiments described by Durkin, et al. (these proceedings), TDM meets all requirements to be X-IFU's backup-readout option. Hybrid C/TDM is another viable option that could save spacecraft resources.

Demonstration of Athena X-IFU Compatible 40-Row Time-Division-Multiplexed Readout.

Time-division multiplexing (TDM) is the backup readout technology for the X-ray Integral Field Unit (X-IFU), a 3,168-pixel X-ray transition-edge sensor (TES) array that will provide imaging spectroscopy for ESA's Athena satellite mission. X-0IFU design studies are considering readout with a multiplexing factor of up to 40. We present data showing 40-row TDM readout (32 TES rows + 8 repeats of the last row) of TESs that are of the same type as those being planned for X-IFU, using measurement and analysis parameters within the ranges specified for X-IFU. Singlecolumn TDM measurements have best-fit energy resolution of (1.91 ± 0.01) eV for the Al Kα complex (1.5 keV), (2.10 ± 0.02) eV for Ti Kα (4.5 keV), (2.23 ± 0.02) eV for Mn Kα (5.9 keV), (2.40 ± 0.02) eV for Co Kα (6.9 keV), and (3.44 ± 0.04) eV for Br Kα (11.9 keV). Three-column measurements have best-fit resolution of (2.03 ± 0.01) eV for Ti Kα and (2.40 ± 0.01) eV for Co Kα. The degradation due to the multiplexed readout ranges from 0.1 eV at the lower end of the energy range to 0.5 eV at the higher end. The demonstrated performance meets X-IFU's energy-resolution and energy-range requirements. True 40-row TDM readout, without repeated rows, of kilopixel scale arrays of X-IFU-like TESs is now under development.

Microwave SQUID Multiplexer Demonstration for Cosmic Microwave Background Imagers.

Key performance characteristics are demonstrated for the microwave SQUID multiplexer (µmux) coupled to transition edge sensor (TES) bolometers that have been optimized for cosmic microwave background (CMB) observations. In a 64-channel demonstration, we show that the µmux produces a white, input referred current noise level of [Formula: see text] at -77 dB microwave probe tone power, which is well below expected fundamental detector and photon noise sources for a ground-based CMB-optimized bolometer. Operated with negligible photon loading, we measure [Formula: see text] in the TES-coupled channels biased at 65% of the sensor normal resistance. This noise level is consistent with that predicted from bolometer thermal fluctuation (i.e. phonon) noise. Furthermore, the power spectral density is white over a range of frequencies down to ~ 100 mHz, which enables CMB mapping on large angular scales that constrain the physics of inflation. Additionally, we report cross-talk measurements that indicate a level below 0.3%, which is less than the level of cross-talk from multiplexed readout systems in deployed CMB imagers. These measurements demonstrate the µmux as a viable readout technique for future CMB imaging instruments.

Upper-limb function in Australian children with traumatic brain injury: A controlled, prospective study.

OBJECTIVE: To describe upper-limb function in children with mild and severe traumatic brain injury (TBI), by using both quantitative and qualitative measures. DESIGN: Controlled, prospective cohort study with assessment points initially, at 6 months, and at 2 years after TBI. SETTING: A tertiary pediatric trauma center in Australia. PATIENTS: Fifty-one children, ranging in age up to 14 years, who were consecutive admissions with TBI. On the basis of initial and persisting abnormal coma score and persistence of posttraumatic amnesia, they were assigned to either a mild (n = 26) or a severely injured (n = 25) group. Thirty children admitted with non-TBI trauma were recruited as a control group. MAIN OUTCOME MEASURES: Quantitative measures included Bruininks-Oseretsky Test of Motor Proficiency and Peabody Developmental Motor Scales. Qualitative measures included Brunnstrom Recovery Stages (adapted), categoric scales of muscle tone, grasp used when handwriting, quality of writing product, bilateral activity, and splint use. RESULTS: There was little difference between the groups on the standardized assessments for subjects who could complete the tests. Qualitative measures showed the severe TBI group to have more difficulties with gross arm control, hand control, and hand function. CONCLUSION: Children with severe TBI experience more and persisting difficulties with upper-limb function. It is essential to include both quantitative and qualitative measures in this type of research.

Serum albumin binds beta- and alpha-monoolein in vitro.

We investigated the interaction of bovine serum albumin (BSA) and monoolein (MO) and estimated the number of BSA binding sites for the alpha- and beta-isomers of MO. The turbidity of increasing concentrations of aqueous dispersions of alpha-MO and beta-MO in the presence and absence of BSA was measured in triplicate by absorption spectrophotometry. Aqueous dispersions of [13C(1)]MO and [13C(1)]MO/BSA mixtures at molar ratios of 1:1, 3:1 and 5:1 were analyzed in duplicate by [13C]nuclear magnetic resonance (NMR) at pH 7.4 and 36 degrees C. BSA bound significantly more beta-MO than alpha-MO at 15 min: 5.4 +/- 0.42 and 3.3 +/- 0.60 mol MO/mol BSA, respectively (P: < 0.05). [13C]NMR spectra of the 1:1 molar ratio of [13C(1)]MO /BSA exhibited a single carbonyl peak at 175.19 ppm, whereas spectra of 3:1 and 5:1 molar ratios exhibited three peaks between 172 and 174 (ppm), each distinct from carbonyl resonances of either [13C(1)]MO dispersed in water, 176.72 (ppm) or BSA alone. The intensities of individual peaks, but not their chemical shift values, varied between 3:1 and 5:1 molar ratios, indicating that BSA has at least three MO binding sites and may bind up to five molecules of MO per molecule. This study confirms that serum albumin binds MO in vitro and supports the theory that albumin transports monoglycerides produced by lipoprotein lipase hydrolysis of triglyceride.

The heme environment in barley hemoglobin.

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm-1, with relative intensities that are pH sensitive. The 534 cm-1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogen-bonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm-1 is assigned as being associated with the 534 cm-1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm-1, respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm-1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.

Altering hemoglobin levels changes energy status in maize cells under hypoxia.

Nonsymbiotic hemoglobins are broadly present across the plant kingdom; however, the function of these proteins is unknown. Cultured maize cells have been transformed to constitutively express a barley hemoglobin gene in either the sense (HB+) or antisense (HB-) orientation. Hemoglobin protein in the transformed cell lines correspondingly was higher or lower than in wild-type cells under normal atmospheric conditions. Limiting oxygen availability, by placing the cells in a nitrogen atmosphere for 12 hr, had little effect on the energy status of cells constitutively expressing hemoglobin, but had a pronounced effect on both wild-type and HB- cells, where ATP levels declined by 27% and 61%, respectively. Total adenylates in these cells were approximately 35% lower than in HB+ cells. Energy charge was relatively unaffected by the treatment in HB+ and wild-type cells, but was reduced from 0.91 to 0.73 in HB- cells, suggesting that the latter were incapable of maintaining their energy status under the low oxygen regime. Treatment of the cells grown in an air atmosphere with antimycin A gave essentially the same results. It is suggested that nonsymbiotic hemoglobins act in plants to maintain the energy status of cells in low oxygen environments and that they accomplish this effect by promoting glycolytic flux through NADH oxidation, resulting in increased substrate-level phosphorylation. Hypoxic acclimation of plants is an example of this effect in nature. Nonsymbiotic hemoglobins are likely ancestors of an early form of hemoglobin that sequestered oxygen in low oxygen environments, providing a source of oxygen to oxidize NADH to provide ATP for cell growth and development.

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands.

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.

Soybean root nodule acid phosphatase.

Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study, we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 microM, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.

Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.

Partial purification and characterization of maize-leaf pyruvate, orthophosphate dikinase regulatory protein: a low-abundance, mesophyll-chloroplast stromal protein.

The C4 mesophyll-chloroplast stromal enzyme pyruvate, orthophosphate dikinase (PPDK) regulatory protein (RP) catalyzes the activation/dephosphorylation and inactivation/phosphorylation of PPDK. This bifunctional converter enzyme has been partially purified from maize leaf extracts approximately 100-fold to a final specific PPDK-inactivation activity of 3-10 units PPDK/min/mg protein and an overall recovery of 1-5%. Purification of active RP was accomplished by (NH4)2SO4 fractionation and sequential column chromatography on dye-ligand and size-exclusion or FPLC-based anion-exchange matrices. Immunoblot analyses of size-exclusion and anion-exchange fractionated preparations indicate that NADP-malate dehydrogenase (MDH) and ribulose-bisphosphate carboxylase/oxygenase are major contaminants, respectively. During sequential dye-ligand and anion-exchange chromatography, RP activity copurifies with a approximately 48-kDa polypeptide on silver-stained denaturing gels. Both RP activities are readily detected in maize mesophyll-chloroplast stromal extracts, but unlike stromal PPDK and NADP-MDH, the putative, approximately 48-kDa RP polypeptide is barely detectable in silver-stained gels. Our collective data indicate that RP constitutes < 0.04% of the total soluble maize-leaf protein and < 1% of its target enzyme, contrary to a previous report. PPDK-inactivation and -activation activities are quantitatively equivalent in light- and dark-adapted maize-leaf tissue rapidly extracted and assayed at either pH 7.0 or 8.0, suggesting that the opposing RP activities are not regulated differentially by reversible covalent modification or pH effects.

An engineered change in the L-malate sensitivity of a site-directed mutant of sorghum phosphoenolpyruvate carboxylase: the effect of sequential mutagenesis and S-carboxymethylation at position 8.

A recombinant, site-directed mutant form of sorghum phosphoenolpyruvate carboxylase (PEPC), in which the phosphorylatable serine residue (Ser-8) was changed to cysteine (S8C), was chemically modified by iodoacetic acid and iodoacetamide for the purpose of testing the effect of introducing a negative charge at position 8. S-Carboxymethylation of the Cys-8 enzyme by iodoacetic acid decreased its sensitivity to L-malate from an I0.5 (50% inhibition) value of 0.12 to 0.35 mM at pH 7.3 when the active-site domain was protected during modification by the substrate phosphoenolpyruvate (PEP). In contrast, neither S-carboxymethylation of the wild-type enzyme nor modification of the mutant enzyme by iodoacetamide caused any change in the enzyme's sensitivity to L-malate. The modified, substrate-protected forms of the Ser-8 and S8C PEPCs had Km(total PEP) and Vmax values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I0.5 value of L-malate have been reported previously for in vitro phosphorylated leaf and recombinant Ser-8 PEPCs, the site-directed mutant Asp-8 enzyme, and C4-leaf PEPC purified from light-adapted sorghum or maize (in vivo phospho-form). Therefore, these data from different but complementary experimental approaches provide convincing evidence that the effect of phosphorylation of Ser-8 on the L-malate sensitivity of sorghum C4-PEPC is caused by the introduction of negative charge into this N-terminal regulatory domain.

Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells.

PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation. Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells. Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM). Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP. By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells. Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate. These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.

Site-directed mutagenesis of the phosphorylatable serine (Ser8) in C4 phosphoenolpyruvate carboxylase from sorghum. The effect of negative charge at position 8.

The properties of the dephospho and in vitro phosphorylated forms of recombinant sorghum phosphoenolpyruvate carboxylase have been compared with those of the authentic dark (dephospho) and light (phospho) leaf enzyme forms and two mutant enzymes in which the phosphorylatable serine residue (Ser8) has been changed by site-directed mutagenesis to Cys (S8C) or Asp (S8D). Kinetic analysis of the purified recombinant, mutant, and leaf enzyme forms at pH 8.0 indicated virtually identical Vmax, apparent Km (phosphoenolpyruvate), and half-maximal activation (glucose 6-P) values of about 44 units/mg, 1.1 mM, and 0.23 mM, respectively. In contrast, the Ser8, S8C, and dark leaf enzymes were about 3-fold more sensitive to inhibition by L-malate at pH 7.3 than the Ser8-P, S8D, and light leaf enzyme forms. These comparative results indicate that: (i) Ser8 is an important determinant in the regulation of sorghum phosphoenolpyruvate carboxylase activity by negative (L-malate), but not positive (glucose 6-phosphate) metabolite effectors, (ii) phosphorylation of this target residue can be functionally mimicked by Asp, but not Cys, and (iii) negative charge contributes to the effect of regulatory phosphorylation on this C4-photosynthesis enzyme.

Phosphate-starvation response in plant cells: de novo synthesis and degradation of acid phosphatases.

Induction of phosphatase activity is an important component of the plant cell response to phosphate deficiency. Suspension cell cultures of Brassica nigra contain two major inducible acid phosphatase (APase) isozymes; vacuolar phosphoenolpyruvate (PEP) APase and cell wall nonspecific APase. Polyclonal antibodies raised against purified PEP-APase crossreacted specifically with both isozymes. Furthermore, anti-(PEP-APase) IgG detected proteins from a wide range of higher plants, suggesting that the major plant APase isozymes have diverged from a common ancestral form. Quantification on immunoblots indicated that in B. nigra suspension cells experiencing transition from Pi sufficiency to deficiency or vice versa, the amount of total antigenic APase protein correlated closely with total enzyme activity. This was also shown in intact plant roots. Therefore, the activity was governed by the synthesis and degradation of APases. Increases in the amounts of both major APase isozymes occurred simultaneously following Pi deprivation of B. nigra suspension cells, suggesting the involvement of a common regulatory mechanism.

Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: comparison with phosphoenolpyruvate phosphatase.

An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.

Response to Phosphate Deprivation in Brassica nigra Suspension Cells : Enhancement of Intracellular, Cell Surface, and Secreted Phosphatase Activities Compared to Increases in Pi-Absorption Rate.

Suspension cells of Brassica nigra responded to Pi deprivation by increasing their potential for Pi influx and by raising the active levels of intracellular, cell surface, and secreted acid phosphatases. These responses, however, were temporally distinct. Phosphate influx capacity increased 15-fold in parallel to a 10-fold decrease in endogenous Pi during 7 days of culture in basal growth medium. In contrast, intracellular and cell surface phosphatase activities changed only after alterations in cellular phosphorus status had been in place for a number of days. Even in nutrient sufficient cells the secretion of phosphatase remained relatively high as did the activities of the other phosphatases. The cell surface acid phosphatase had a K(m) of approximately 10 times that of the influx process and molybdate was a much stronger inhibitor of this phosphatase activity. From these results it appears that Pi absorption and the production or activation of phosphatases are regulated in a distinct manner. In addition, Pi uptake into Brassica nigra cells does not appear to directly involve the cell surface phosphatase under Pi-deficient conditions.

Phosphate Starvation Inducible ;Bypasses' of Adenylate and Phosphate Dependent Glycolytic Enzymes in Brassica nigra Suspension Cells.

When Brassica nigra leaf petiole suspension cells were subjected to 7 days of inorganic phosphate (Pi) starvation the extractable activity of: (a) pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate phosphatase, and phosphoenolpyruvate carboxylase increased at least fivefold, (b) phosphorylating NAD-glyceraldehyde 3-phosphate dehydrogenase decreased about sixfold, and (c) ATP:fructose 6-phosphate 1-phosphotransferase, 3-phosphoglycerate kinase, pyruvate kinase, or NAD malic enzyme was not altered. Pi deprivation also resulted in significant reductions in extractable levels of Pi, ATP, ADP, fructose 2,6-bisphosphate, and soluble protein, but caused a sixfold elevation in free amino acid concentrations. No change in inorganic pyrophosphate concentration was observed following Pi starvation. It is hypothesized that pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion.

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells.

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.