Plant cytosolic pyruvate kinase: a kinetic study.

The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP. Mg2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc.

A fluorescence study of ligand-induced conformational changes in cytosolic fructose-1,6-bisphosphatase from germinating castor oil seeds.

The intrinsic fluorescence of homogeneous castor oil seed cytosolic fructose-1,6-bisphosphatase (FBPasec) was used as an indicator of conformational changes due to ligand binding. Binding of the substrate and the inhibitor fructose-2,6-bisphosphate (F-2,6-P2) was quantitatively compared to their respective kinetic effects on enzymatic activity. There are two distinct types of substrate interaction with FBPasec, corresponding to catalytic and inhibitory binding, respectively. Inhibitory substrate binding shares several characteristics with F-2,6-P2 binding which indicates that both ligands bind at the same site. However, F-2,6-P2 does not prevent fluorescence transitions attributed to catalytic substrate binding. The marked synergistic inhibition of FBPasec by AMP and F-2,6-P2 appears to arise via AMP's promotion of F-2,6-P2 binding. Based on the X-ray crystal structure of porcine kidney FBPase our modelling studies suggest the existence of a distinct F-1,6-P2/F-2,6-P2 inhibitory binding site which partially overlaps with the enzyme's catalytic site. We propose that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P2 and F-2,6-P2 to this common inhibitory binding site.

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation.

Plants respond adaptively to orthophosphate (Pi) deprivation through the induction of alternative pathways of glycolysis and mitochondrial electron transport. These respiratory bypasses allow respiration to proceed in Pi-deficient plant cells because they negate the necessity for adenylates and Pi, both pools of which are severely depressed following nutritional Pi starvation.

Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol.

Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo.

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds.

Plastid pyruvate kinase (PKp) activity and anti-(castor oil seed [COS] PKp) immunoglobulin G immunoreactive polypeptides were recovered in the stroma but not from envelope membranes of purified COS leukoplasts that had been subfractionated by sucrose density gradient centrifugation. The PKp was highly purified from isolated leukoplasts using anion-exchange and ADP-agarose chromatographies. Proteolysis of PKp was almost entirely eliminated by including 2,2[prime]-dipyridyl disulfide in purification buffers. The final preparation contained 63.5-kD ([alpha] subunit) and 54-kD ([beta] subunit) polypeptides that stained for protein and cross-reacted with anti-(COS PKp) immunoglobulin G with similar intensities. These two polypeptides co-eluted following gel-filtration chromatography and co-migrated during nondenaturing isoelectric focusing-polyacrylamide gel electrophoresis. The enzyme's native Mr was estimated to be 334,000. This PKp thus appears to exist as an [alpha]3[beta]3-heterohexamer. Comparison of the respective N-terminal sequences of the [alpha] and [beta] subunits with the deduced amino acid sequences for several PKp cDNAs indicated that (a) the [alpha] and [beta] subunits are encoded by COS genes previously designated as PKpA and PKpG, respectively, and (b) respective transit peptides of 4.8- and 5.5-kD are cleaved from the [alpha] and [beta] subunit preproteins following their translocation into the leukoplast.

The Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica nigra Seedlings.

The development of Brassica nigra seedlings over 20 d of growth was disrupted by the fungicide phosphonate (Phi) in a manner inversely correlated with nutritional inorganic phosphate (Pi) levels. The growth of Pi-sufficient (1.25 mM Pi) seedlings was suppressed when 10, but not 5, mM Phi was added to the nutrient medium. In contrast, the fresh weights and root:shoot ratios of Pi-limited (0.15 mM) seedlings were significantly reduced at 1.5 mM Phi, and they progressively declined to about 40% of control values as medium Phi concentration was increased to 10 mM. Intracellular Pi levels generally decreased in Phi-treated seedlings, and Phi accumulated in leaves and roots to levels up to 6- and 16-fold that of Pi in Pi-sufficient and Pi-limited plants, respectively. Extractable activities of the Pi-starvation-inducible enzymes phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase were unaltered in Pi-sufficient seedlings grown on 5 or 10 mM Phi. However, when Pi-limited seedlings were grown on 1.5 to 10 mM Phi (a) the induction of phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase activities by Pi limitation was reduced by 40 to 90%, whereas (b) soluble protein concentrations and the activities of the ATP-dependent phosphofructokinase and pyruvate kinase were unaffacted. It is concluded that Phi specifically interrupts processes involved in regulation of the Pi-starvation response in B. nigra.

Purification and Characterization of a Potato Tuber Acid Phosphatase Having Significant Phosphotyrosine Phosphatase Activity.

The major acid phosphatase (APase) from potato (Solanum tuberosom L. cv Chiefton) tubers has been purified 2289-fold to near homogeneity and a final O-phospho-L-tyrosine (P-Tyr) hydrolyzing specific activity of 1917 [mu]mol Pi produced min-1 mg-1 of protein. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resolved a single protein-staining band that co-migrated with APase activity. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, glycosylated polypeptides of 57 and 55 kD were observed. The two polypeptides are immunologically closely related, since both proteins cross-reacted on immunoblots probed with rabbit anti-(Brassica nigra APase) immunoglobulin G. Immunoblotting studies revealed that the 55-kD subunit did not arise via proteolytic cleavage of the 57-kD subunit after tissue extraction. The native molecular mass was approximately 100 kD, suggesting that the holoenzyme could exist as either a homodimer or a heterodimer. The enzyme displayed a pH optimum of 5.8, was activated 40% by 4 mM Mg2+, and was potently inhibited by molybdate, vanadate, and ZnCl2. The final preparation displayed the highest activity and specificity constant with P-Tyr, but also dephosphorylated other phosphomonoesters including p-nitrophenylphosphate, O-phospho-L-serine, phosphoenolpyruvate, PPi, and ATP. Antibodies to P-Tyr were used to demonstrate that several endogenous phosphotyrosylated tuber polypeptides could serve as in vitro substrates for the purified APase. Although the precise physiological significance of the potato APase's substantial in vitro activity with P-Tyr remains obscure, the possibility that this APase may function to dephosphorylate certain protein-located P-Tyr residues in vivo is suggested.

Phosphorus nutrition in Proteaceae and beyond.

Proteaceae in southwestern Australia have evolved on some of the most phosphorus-impoverished soils in the world. They exhibit a range of traits that allow them to both acquire and utilize phosphorus highly efficiently. This is in stark contrast with many model plants such as Arabidopsis thaliana and crop species, which evolved on soils where nitrogen is the major limiting nutrient. When exposed to low phosphorus availability, these plants typically exhibit phosphorus-starvation responses, whereas Proteaceae do not. This Review explores the traits that account for the very high efficiency of acquisition and use of phosphorus in Proteaceae, and explores which of these traits are promising for improving the phosphorus efficiency of crop plants.

Evidence for an interaction between cytosolic aldolase and the ATP-and pyrophosphate-dependent phosphofructokinases in carrot storage roots.

Immunoaffinity chromatography was employed to identify potential plant cytosolic aldolase (ALDc) binding proteins. A clarified homogenate of carrot storage root was chromatographed on a column of protein-A-Sepharose that had been covalently coupled to anti-(carrot root ALDc) immunoglobulin G. The column was washed with phosphate-buffered saline (PBS), followed by step-wise elution with increasing concentrations of NaCl in PBS. Several proteins were eluted following application of the salt gradient. Western blotting identified the major eluting proteins to be the PPi-dependent phosphofructokinase (PFP) and the cytosolic form of the ATP-dependent phosphofructokinase (PFKc), enzymes that are metabolically sequential to ALDc. The results suggest that ALDc may specifically interact with PFP and PFKc in carrots.

Effect of polyethylene glycol on the activity, intrinsic fluorescence, and oligomeric structure of castor seed cytosolic fructose-1,6-bisphosphatase.

The effect of polyethylene glycol (PEG) on the activity, intrinsic fluorescence, and oligomeric structure of homogeneous cytosolic fructose-1,6-bisphosphatase (FBPasec) from endosperm of germinating castor oil seeds has been examined. Increasing the PEG concentration in the FBPasec reaction mixture elicited a progressive 3-fold decrease in the enzyme's Km for fructose-1,6-P2. The presence of PEG also: (i) increased the extent of FBPasec inhibition by high levels of fructose-1,6-P2, (ii) enhanced the intensity of the enzyme's fluorescence emission spectra, and (iii) prevents dissociation of the active tetrameric native enzyme into inactive lower M(r) forms during gel filtration HPLC. It is concluded that the activity and structure of plant FBPasec is modified by extreme dilution, probably as a result of partial deaggregation of the native tetrameric enzyme.

Upregulation of vacuolar H(+)-translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures.

The influence of phosphate (Pi) deprivation on the vacuolar H(+)-translocating pyrophosphatase (PPiase) and ATPase in tonoplast vesicles from Brassica napus suspension cells was assessed. Pi starvation significantly elevated the ratios of PPi-:ATP-dependent H(+) translocation rate and H(+)-PPiase:H(+)-ATPase hydrolytic activities. These increases were reversed 36 h following resupply of 2.5 mM Pi to the Pi-starved cells. Immunoblotting indicated that Pi starvation also induced a two-fold increase in the amount of H(+)-PPiase protein, whereas the amount of H(+)-ATPase remained unchanged. It is proposed that H(+)-PPiase facilitates the conservation of limited ATP pools, and Pi recycling during Pi stress.

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase.

Leucoplast pyruvate kinase (PK(p); EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4 degrees C. Purified castor seed PK(p) was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK(p)) immunoglobulin G. Proteolytic activity against PK(p) was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4 degrees C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK(p). The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nalpha-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK(p). This endopeptidase is present throughout all stages of endosperm development. Its PK(p)-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4 degrees C for up to 21 hours, no degradation of PK(p) was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK(p) remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK(p).

Purification of pyruvate kinase from germinating castor bean endosperm.

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.

Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf.

1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of cytosolic aldolase from carrot storage root.

A single fructose-1,6-bisphosphate (FBP) aldolase has been detected in extracts from carrot storage roots (Daucus carota L.). The enzyme was purified 850-fold to electrophoretic homogeneity and a final specific activity of 26.3 mumols of FBP utilized/min per mg of protein. SDS/PAGE of the final preparation revealed a single protein-staining band of 40 kDa. The native molecular mass was determined by analytical gel filtration to be 159 kDa, indicating that the enzyme is a homotetramer. Denaturing isoelectric focusing revealed two predominant protein-staining bands, with pI values of 5.6 and 5.7. The enzyme is a class I aldolase, since EDTA or metal ions had no effect on its activity. The enzyme was relatively heat-stable, had an activation energy (Ea) of 68.3 kJ.mol-1, and had an absorption coefficient of 8.08 x 10(4) M-1.cm-1 at 280 nm. Km values for FBP and sedoheptulose 1,7-bisphosphate (SBP) were both determined to be 6 microM (pH optima 7.4). The specificity constant with FBP was 2.6 times that obtained with SBP. Ribose 5-phosphate, 6-phosphogluconate, MgAMP, glucose 1-phosphate and phosphoenolpyruvate (PEP) were inhibitors. PEP was a mixed-type inhibitor with respect to FBP (Ki = 3.2 mM, K'i = 5.1 mM). No activators were found. Rabbit anti-(carrot aldolase) polyclonal antibodies immunoprecipitated the activity of both carrot root aldolase and spinach leaf cytosolic aldolase, but not that of spinach leaf plastid aldolase. Western-blot analysis also revealed cross-reactivity with cytosolic, but not plastid, spinach leaf aldolase, indicating that the single carrot root aldolase is cytosolic.

Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans.

The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP.

Binding of glycolytic enzymes to a particulate fraction in carrot and sugar beet storage roots : dependence on metabolic state.

Numerous studies have demonstrated a rapid increase in the respiration rate during aging of slices of tuber and storage roots. To determine the molecular mechanisms of this phenomenon, the role of enzyme binding to the subcellular particulate fraction has been assessed in carrot (Daucus carota L.) and sugar beet (Beta vulgaris L.). Soluble versus particulate fractions were separated by centrifugation at 16,000g and both fractions assayed for the activities of six glycolytic enzymes. Preparations from sliced and aged tissues showed elevated percentages of five enzymes associated with the particulate fraction as compared with controls. The stimulation of respiration which occurs during aging of underground storage organ slices may result, in part, from an association of enzymes with the particulate fraction of the cell promoting an elevated glycolytic rate.

Purification and properties of aerobic and anoxic forms of pyruvate kinase from the hepatopancreas of the channelled whelk, Busycotypus canaliculatum.

Aerobic and anoxic variants of pyruvate kinase (termed PK-aer and PK-anx) from the hepatopancreas of the gastropod mollusc, Busycotypus canaliculatum, were purified to apparent homogeneity with final specific activities of 14 and 2.3 units/mg protein, respectively. Both enzymes were homotetramers of the same molecular weight. The enzymes also showed equivalent affinities for ADP (0.22 mM) and very similar affinities for Mg2+, Mn2+, K+, and NH4+. PK-aer and PK-anx differed strongly, however, in maximal enzyme velocity (Vmax 9-fold higher for PK-aer), in affinity for P-enolpyruvate (PEP0.5 = 0.38 mM for PK-aer and 1.1 mM for PK-anx), and in the effects of activators and inhibitors on the enzymes. PK-aer was much more strongly stimulated by fructose-1,6-P2 and aspartate as activators (a 19- and 32-fold activation of enzyme velocity at subsaturating PEP levels versus only 4.1- and 2.6-fold activation for PK-anx, respectively). K alpha for fructose-1,6-P2 was 3-fold lower (0.16 microM) for PK-aer than for PK-anx (0.48 microM), but K alpha for aspartate was the same for both enzymes (1.5 mM). Activators decreased the PEP0.5 (to 0.05 mM for PK-aer and 0.07 mM for PK-anx), relieved inhibitions by alanine, Mg ATP, ADP, and Pi, and, when added together, showed a strong synergistic activation of PK-aer (but not PK-anx). The kinetic differences between PK-aer and PK-anx are similar to those of the dephosphorylated versus phosphorylated forms of PK from other sources, including those of red muscle PK of B. canaliculatum, and indicate that the change in enzyme form brought about during anaerobiosis may be due to enzyme phosphorylation. The powerful activation of hepatopancreas PK by aspartate is a novel regulatory control of the enzyme. Aspartate is one of the substrates of anaerobic energy production in marine molluscs and its effects on the enzyme may be important in a tissue where inactivation of PK can occur for one of two reasons: anaerobiosis or gluconeogenesis.

Peptide mapping by CNBr fragmentation using a sodium dodecyl sulfate-polyacrylamide minigel system.

The method of peptide mapping by sodium dodecyl sulfate-gel electrophoresis following partial protein fragmentation with cyanogen bromide was adapted for a polyacrylamide minigel system. The combined use of the discontinuous gel electrophoresis system of J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271) and a vertical polyacrylamide minigel system produced the following advantages over other procedures: (a) the ability to resolve cyanogen bromide cleavage fragments over a broad molecular mass range while yielding very sharp protein staining bands; (b) well-defined peptide maps are produced with as little as 2 micrograms of protein; (c) less time is required to perform fragmentation with cyanogen bromide, to equilibrate the gel slices in sodium dodecyl sulfate buffer, as well as to perform the electrophoresis; and (d) the cyanogen bromide fragmentation patterns are highly reproducible.

High-yield purification of potato tuber pyrophosphate: fructose-6-phosphate 1-phosphotransferase.

The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman P11 phosphocellulose, anion-exchange chromatography using Q-Sepharose "Fast Flow," and gel filtration chromatography with Superose 6 "Prep grade." Our procedure resulted in an overall 42% yield and a final specific activity of 87 mumol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken.