Origin of tryptophan in fructose 1,6-bisphosphatase purified from livers of fed rabbits.

Fructose 1,6-bisphosphatase (Fru-P₂ase,EC 3.1.3.11) purified from livers of fed rabbits has been reported to contain tryptophan, which is not present in the enzyme purified from livers of fasted animals. We now show that the tryptophan arises from small amounts of active or inactive rabbit liver aldolase in the Fru-P₂ase preparations. Fru-P₂ase free of tryptophan may conveniently be prepared by raising the temperature of the heat step in the purification procedure to 67 °C.     

Inactivation of fructose 1,6-bisphosphatase by a lysosomal proteinase is reversed by cystamine

Inactivation of rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) by a membrane-bound lysosomal proteinase, designated as cathepsin M, is reversed by incubation with cystamine, which has been shown to form a mixed disulfide derivative with protein SH groups. The native form of Fru-P₂ase is also activated by cystamine; this activated form is resistant to the action of membrane-bound cathepsin M.     

On the nucleotide binding domain of fructose-1,6-bisphosphatase.

Native chicken liver fructose-1,6-bisphosphatase (Fru-P₂ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P₂ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH₄ leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P₂ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P₂ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P₂ase, digestion of pyridoxal-P-Fru-P₂ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, M_r ~ 30,000, was found to contain all of the incorporated pyridoxal-P.     

Rabbit liver fructose-1,6-bisphosphatase: location of an active site lysyl residue in the COOH-terminal fragment generated by a lysosomal proteinase

Digestion of native rabbit liver fructose-1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) with a membrane-bound proteinase from rat liver lysosomes yields a fragment of M_r = 9850. This peptide contains the COOH terminus of the Fru-P₂ase polypeptide chain and also the cyanogen bromide peptide (BrCN5) carrying the active site lysyl residue. The sequence of BrCN5 and its location with respect to the COOH terminus of the polypeptide chain have been determined. The active site lysyl residue is located at approximately residue ?54 from the COOH terminus. The bond hydrolyzed by the lysosomal proteinase is located between residues ?88 and ?89 from the COOH terminus.     

Quantitative estimation of newly synthesized radioactive proteins: use of minicolumns of antibody embedded in agarose gel or immobilized on Sepharose.

Immunochemical methods for the quantitative determination of radioactive proteins synthesized by chick embryo hepatocyte cultures are described. Cell culture medium containing secreted labeled serum proteins was concentrated and applied to agarose gels containing rabbit anti-chicken serum. Nonspecific binding in control gels was reduced to less than 2% of applied counts under conditions where more than 60% of the nondialyzable counts were precipitated in the presence of antibody. Labeled cytosol fructose-1,6-bisphosphatase (Fru-P₂ase) from cell sonicates was selectively adsorbed onto columns containing Sepharose-immobilized anti-chicken liver Fru-P₂ase. Radioactivity bound on these columns was eluted with 1% sodium dodecyl sulfate for electrophoretic analysis. Addition of dialyzed horse serum was used in both cases to minimize nonspecific binding. These techniques provide simple alternatives to direct immunoprecipitations in solution where nonspecific binding of radioactivity may be unacceptably high.     

The carboxy-terminal amino acid sequence of rabbit liver fructose 1,6-bisphosphatase

A peptide derived from the COOH-terminus of rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) has been isolated and its amino acid sequence determined. The COOH-terminus is lysine, but some preparations contain COOH-terminal alanine or lysyl lysine. This region of the protein appears to be susceptible to modification by the action of an endogenous peptidyldipeptidase.     

Rabbit liver fructose 1,6-bisphosphatase: labeling of the active and allosteric sites with pyridoxal 5-phosphate and sequence of a nonapeptide from the active site

The active and allosteric sites of fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) were labeled by reaction with pyridoxal phosphate and sodium borohydride in the presence of the allosteric inhibitor AMP or the substrate, Fru-P₂, respectively. Modification of the active site results in loss of activity. Modification of the allosteric site decreases the sensitivity of the enzyme to inhibition by AMP and alters its ability to bind to blue dextran-Sepharose. The allosteric and active sites have been located on different cyanogen bromide peptides; the sequence of a nonapeptide from the active site is (H)GlyLysLeuArgLeuLeu TyrGluCys(OH). The lysyl residue is modified by pyridoxal phosphate.     

The effect of Zn2+ on the inhibition of Fru-P2ase by AMP.

In the absence of chelating agents, the sensitivity of rabbit liver fructose 1,6-bisphosphatase to inhibition by AMP is increased by approximately 10-fold, apparently related to the presence of bound Zn²⁺. At pH 7.5, 10 μM AMP causes virtually complete inhibition and nearly full activity is restored by the addition of chelating agents such as EDTA or histidine. These properties provide a mechanism for the induction of Fru-P₂ase activity under gluconeogenic conditions.     

Properties of a fructose 1,6-bisphosphatase converting enzyme in rat liver lysosomes.

An enzyme present in rat liver lysosomes catalyzes the conversion of neutral rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) to a form having maximum activity at pH 9.2. The converting enzyme is partly released when lysosomes are subjected to a single freeze-thaw cycle, but a significant fraction tends to remain with the lysosomal membrane fraction even after repeated freezing and thawing. After repeated freezing and thawing hexosaminidase and cathepsin D are also partly membrane-bound, but cathepsins A, B, and C are completely solubilized. The membrane-bound enzymes, unlike those in intact lysosomes, are not cryptic. The converting enzyme activity is inactivated by phenylmethanesulfonyl fluoride, and is almost completely inactive after exposure to iodoacetic acid or tosylamido-2-phenylethyl and N-α-tosyl lysyl chloromethyl ketones. Unlike cathepsin B, it is not inhibited by leupeptin. Converting enzyme is unstable above pH 6.5, and this property also serves to distinguish it from cathepsins B and D. The results suggest that the converting enzyme is not identical to any of the well-characterized cathepsins.     

Differential Localization of Fraction I Protein between Chloroplast Types

The soluble proteins of C₃ and C₄ mesophyll chloroplasts and C₄ bundle sheath extracts have been analyzed by gel electrophoresis for fraction I protein. Gel scans of soluble protein from C₄ bundle sheath extracts and C₃ mesophyll chloroplasts showed typical fraction I protein peaks that could be identified by ribulose diphosphate carboxylase activity. No such peak was observed for C₄ mesophyll chloroplasts, which also lacked both large and small subunits of ribulose diphosphate carboxylase on sodium dodecyl sulfate gels. The absence of fraction I protein in these chloroplasts was reflected in the soluble protein to chlorophyll ratios, which were roughly 3-fold lower than the ratio obtained for C₃ chloroplasts. The carboxylating enzyme in C₄ mesophyll cells, phosphoenolpyruvate carboxylase, was found to be a major protein in the cytoplasm of C₄ mesophyll protoplasts, and had higher mobility than fraction I protein.     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

Specific Labeling of the Phosphate Translocator in C(3) and C(4) Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2' -Disulfo-4,4' -Diisothiocyano] Diphenylethane)

The phosphate translocator protein of C₃ and C₄ mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2′ -disulfo-4,4′ -diisothiocyano) diphenylethane ([³H]₂-DIDS). Intact mesophyll chloroplasts were isolated from the C₃ plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C₄ plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 μm [³H]₂-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [³H]₂-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C₃ phosphate translocator) for both C₃ and C₄ chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C₃ and C₄ phosphate translocator protein(s).     

Occurrence and localization of an uptake hydrogenase in the filamentous heterocystous cyanobacteriumNostoc PCC 73102

Summary Free-living nitrogen-fixingNostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycadMacrozamia sp., were examined for the presence of an uptake hydrogenase (H₂ase) enzyme. In vivo and in vitro hydrogen uptake measurements were used to study activities and SDS-PAGE and Western immunoblots to reveal occurrence of the hydrogenase protein. Also, transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of H₂ase in theNostoc cells. In vivo measurements demonstrated an active uptake of hydrogen in both light and darkness. Light stimulated in vivo hydrogen uptake with approximately 100%, and this was further doubled by increasing the pH₂, from 56 to 208 M H₂. An in vitro hydrogen uptake of 1.1 mol H₂/ mg (protein)/h was observed when using phenazinemethosulphate as e^–-acceptor. Western immunoblots revealed that a polypeptide with a molecular weight of about 55 kDa was immunologically related to uptake H₂ase holoenzyme purified fromAlcaligenes latus. Immunolocalization demonstrated that the H₂ase protein was located both in heterocysts and vegetative cells. A higher specific labeling was associated with the cytoplasmic membranes where the vegetative cells are in contact with each other and where they actually are dividing into two vegetative cells. Using the particle analysis of an image processor, approximately equal H₂ase-gold labeling per cell area was observed in the nitrogen-fixing heterocysts compared to the photosynthetic vegetative cells. This study also shows that there was no correlation between presence of phycoerythrin and uptake H₂ase activity.Abbreviations H₂ase hydrogenase - IgG immunoglobulin G     

Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation : REGULATION BY PRIMER, ADENYLATES, FERREDOXIN, AND PYRIDINE NUCLEOTIDES

A particulate preparation (MgP) capable of photosynthetic CO₂ assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl₂ buffered with Tricine at pH 8.5. This CO₂ assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO₂ fixation by MgP were similar to those found with whole chloroplasts.     

A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii

The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO₂. Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO₂-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO₂-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO₂-grown C. reinhardtii cells.     

Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H₂O₂. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H₂O₂, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN₃. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H₂O₂. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.     

Impact of elevated CO2 concentration on ultrastructure of pericarp and composition of grain in three Triticum species of different ploidy levels

Triticum species of three ploidies were grown under ambient (375 μl/l) or elevated (FACE, 550 μl/l) CO₂ concentration [CO₂] to evaluate their response to CO₂ enrichment. The consistent effect of elevated CO₂ was an increase in concentration of starch and decrease in concentration of protein in the grain. Transmission electron micrographs revealed an increase in width and area of chloroplasts, and change in shape from elliptical in ambient to round in elevated [CO₂]. There was a corresponding increase in starch grain size and number in chloroplasts. The large starch grains distributed among thylakoids resulted in separation and distortion of internal membrane system in chloroplasts. The level of response was different in species of different ploidy levels. Maximum increase in starch concentration, and least decrease in protein concentration, was observed in Triticum dicoccoides, which also proved the most suitable species in terms of C:N ratio.     

Free flow electrophoresis of chloroplasts

Highly purified intact chloroplasts were isolated from spinach (Spinacia oleracea L.) leaves by free flow electrophoresis. Morphological and biochemical studies showed that the fraction enriched in intact chloroplasts has a higher protein to chlorophyll ratio and a higher linolenic acid content than the broken organelles of the other fraction. The intact chloroplasts prepared by electrophoresis retained their capacity for CO₂ fixation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that this fraction was rich in stroma and lamellae proteins. Free flow electrophoresis, which separates organelles and molecules according to their surface charges, is a good technique for producing purified chloroplasts with complete physiological activities.     

Protein synthesis in isolated spinach chloroplasts: comparison of light-driven and ATP-driven synthesis

A comparison has been made of the optimal concentrations of Mg²⁺ and K⁺ ions necessary for both light-driven protein synthesis in intact spinach chloroplasts and for ATP-driven protein synthesis in broken chloroplasts, and the products of the two systems have been compared by polyacrylamide gel electrophoresis. Light-driven incorporation of amino acids into polypeptides in intact chloroplasts assayed in buffer systems containing sucrose or sorbitol as the osmoticum is inhibited by the addition of Mg²⁺, the effect being most marked at low concentrations (less than 40 mm) of KCl. On the other hand, chloroplasts suspended in 0.2 m KCl as osmoticum require Mg²⁺ (3 mm) for optimal light-driven protein-synthesizing activity. Incorporation of amino acids by broken chloroplasts in the dark, supplemented with ATP and GTP, requires 9 mm Mg²⁺ for maximum activity. A requirement for monovalent cations is best filled by K⁺ (approx 30 mm) in the case of the light-driven, intact chloroplast system whereas, in the ATP-driven, broken chloroplast system, NH_4⁺ (approx 80 mm) gave the highest activity.Autoradiographs of Na dodecyl sulfate-polyacrylamide gels of the products from both the light-driven, intact chloroplasts and from the ATP-driven, broken chloroplasts reveal qualitatively similar patterns. There are at least four radioactive polypeptides in the soluble protein fraction the dominant product being coincident with the large subunit of Fraction 1 protein. In the membrane fraction at least nine discrete products can be resolved.