Isolation and characterization of androgen-dependent non-histone chromosomal protein from dorsolateral prostate of rats

Rat prostate contains a unique androgen-dependent non-histone protein (Matuo et al. (1)). The non-histone protein was isolated in homogeneous form by extraction of nuclei from the dorsolateral prostate with 0.35 M NaCl in the presence of 1 mM PMSF and chromatography on a CM-Sepharose column. The final fraction was greater than 98% pure as judged by electrophoreses in SDS- and acid/urea-gels. The purified protein had a molecular weight of approximately 20,000, and an isoelectric point of approximately 11.5. Its absorption peak was at 276 nm and A(1%, 276nm)=9.3. The protein is characterized by the absence of cysteine, histidine and tryptophan, and by the high content of methionine, tyrosine and phenylalanine.     

A new procedure for the purification of spinach leaf photosynthetic fructose-1,6-bisphosphatase by affinity chromatography on mercaptoethylamine-Sepharose

A new purification procedure for spinach leaf fructose-1,6-bisphosphatase is proposed, which includes the use of affinity chromatography on mercaptoethylamine-Sepharose. A homogeneous preparation of the enzyme can be obtained in 48 hr, with a specific activity of 67 U/mg and a yield of 23%. The method may also be useful for the purification of other thioredoxin-activated chloroplast enzymes.     

Glu 69 is essential for the high sensitivity of muscle fructose-1,6-bisphosphatase inhibition by calcium ions

Muscle fructose-1,6-bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52-72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu(69)/Gln were employed. The chemical modification of Lys(71-72) and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu(69)-->Gln resulted in a 500-fold increase of muscle isozyme I(0.5) vs. calcium ions. These results demonstrate the key role that the 52-72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.     

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.     

Changes in subcellular localization of fructose 1,6-bisphosphatase during differentiation of isolated muscle satellite cells

Subcellular localization of FBPase, a regulatory enzyme of glyconeogenesis, was examined inside dividing and differentiating satellite cells from rat muscle. In dividing myoblasts, FBPase was located in cytosol and nuclei. When divisions ceased, FBPase became restricted to the cytosolic compartment and finally was found to associate with the Z-lines, as in adult muscle. Moreover, a 12-fold decrease was observed in the number of FBPase-positive nuclei associated with muscle fibres of adult rat, as compared with young muscle, possibly reflecting the reduction in number of active satellite cells during muscle maturation. The data might suggest that FBPase participates in some nuclear processes during development and regeneration of skeletal muscle.     

Overexpression in Escherichia coli and characterization of the chloroplast fructose-1,6-bisphosphatase from wheat

An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones. The overexpressed wheat FBPase is soluble, fully active, and heat stable. It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria. Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min. mg. The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4).     

Chloroplast fructose-1,6-bisphosphatase from Oryza differs in salt tolerance property from the Porteresia enzyme and is protected by osmolytes

Salinity exerted a distinctly differential effect on fructose-1,6-bisphosphatase (EC. 3.1.3.11) isolated from salt-sensitive and salt-tolerant rice (Oryza sativa) varieties. Cytosolic and chloroplastic isoforms of the enzyme from salt-sensitive rice seedlings exhibited decreased catalytic activity during growth in the presence of salt. Furthermore, chloroplastic fructose 1,6-bisphosphatase purified from salt-sensitive (O. sativa cv. IR26) and from the wild halophytic rice Porteresia coarctata differed in their in vitro salt tolerance property although they exhibited otherwise identical biochemical and immunological properties. This decline in enzyme activity was not correlated with de novo synthesis of the chloroplastic fructose-1,6-bisphosphatase protein in the presence of salt. The inhibitory effect of increasing concentration of NaCl on in vitro enzymatic activity could be prevented by preincubation of the enzyme with a number of osmolytes with an effectiveness in the order polyol>sugars. Further, the intrinsic tryptophan fluorescence of the purified rice enzyme is altered in vitro with increasing NaCl concentration which could be prevented by preincubation with inositol. Purified chloroplastic fructose-1.6-bisphosphatase from P. coarctata however, exhibits no such inhibition of enzyme activity in vitro or alteration in tryptophan fluorescence with increasing NaCl concentration.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Modification of cys-128 of pig kidney fructose 1,6-bisphosphatase with different thiol reagents: Size dependent effect on the substrate and fructose-2,6-bisphosphate interaction

Treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide was shown to abolish the inhibition by fructose 2,6-bisphosphate, which also protected the enzyme against this chemical modification [Reyes, A., Burgos, M. E., Hubert, E., and Slebe, J. C. (1987),J. Biol. Chem. 262, 8451–8454]. On the basis of these results, it was suggested that a single reactive sulfhydryl group was essential for the inhibition. We have isolated a peptide bearing the N-ethylmaleimide target site and the modified residue has been identified as cysteine-128. We have further examined the reactivity of this group and demonstrated that when reagents with bulky groups are used to modify the protein at the reactive sulfhydryl [e.g., N-ethylmaleimide or 5,5-dithiobis-(2-nitrobenzoate)], most of the fructose 2,6-bisphosphate inhibition potential is lost. However, there is only partial or no loss of inhibition when smaller groups (e.g., cyanate or cyanide) are introduced. Kinetic and ultraviolet difference spectroscopy-binding studies show that the treatment of fructose 1,6-bisphosphatase with N-ethylmaleimide causes a considerable reduction in the affinity of the enzyme for fructose 2,6-bisphosphate while affinity for fructose 1,6-bisphosphate does not change. We can conclude that modification of this reactive sulfhydryl affects the enzyme sensitivity to fructose 2,6-bisphosphate inhibition by sterically interfering with the binding of this sugar bisphosphate, although this residue does not seem to be essential for the inhibition to occur. The results also suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate may interact with the enzyme in a different way.     

Inhibition of protein phosphorylation by chloroquine

The rapid phase of fructose-1,6-bisphosphatase (FBPase) inactivation following glucose addition to starved yeast cells [reported previously] is inhibited on addition of 10 mM chloroquine (CQ) at about pH 8. This inhibition of inactivation was shown to be due to the prevention of phosphorylation of the enzyme. CQ was also found to inhibit general protein phosphorylation in the yeast cells. Glycolysis, as observed by changes in intracellular glucose-6-phosphate and extracellular glucose and ethanol concentrations, was shown to be significantly inhibited in cells treated with CQ. Similarly, a decrease in ATP concentrations was observed. However, during the early stages of phosphorylation of FBPase, levels of ATP were similar in cells containing CQ as in those without CQ. Thus, decrease in ATP levels is not thought to be significantly responsible for the inhibition of protein phosphorylation. However, the phosphorylating activity of cyclic AMP-dependent protein kinases is inhibited in vitro by relatively low concentrations of CQ. Thus, prevention of protein phosphorylation by CQ is believed to be due to inhibition of protein kinases in yeast cells.Abbreviations FBPase fructose-1,6-bisphosphatase - CQ chloroquine - SDS sodium dodecyl sulfate - G6P glucose-6-phosphate - TCA trichloroacetic acid     

Purification and characterization of an ATP-dependent protein kinase from Streptococcus faecalis

Abstract An enzyme catalyzing the ATP-dependent phosphorylation of HPr of the bacterial phosphotransferase system has been purified from Streptococcus faecalis . Size exclusion chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gels revealed an M _r of 65000. Beside HPr of S. faecalis the protein kinase also phosphorylates HPr of Streptococcus lactis, Streptococcus pyogenes, Bacillus subtilis and Streptococcus aureus , but not HPr of Escherichia coli . The kinase is largely inhibited by P_i and EDTA. Mg²⁺ and Mn²⁺ could overcome inhibition by EDTA. 2-Phosphoglycerate and glucose-6-phosphate, previously reported to stimulate kinase activity in crude extracts, had no effect on the purified enzyme. Fructose-1,6-diphosphate stimulated the protein kinase.     

Beta-D-Galactoside transport in Escherichia coli: Mr determination of the transport protein in organic solvent

Parathyroid hormone stimulates [U-14C]glucose incorporation into glycogen of cultured osteoblast-like calvaria cells. This effect is detectable only several hours after the addition of PTH and it is mimicked by dibutyryl cyclic AMP. In contrast to insulin (in pharmacological concentrations), PTH enhances glycogen formation only in calvaria cells, but not in fibroblasts. Insulin-like growth factor I in physiological concentrations promotes glycogen-synthesis shortly after addition.     

Effect of alcohols on the association of photosynthetic fructose-1,6-bisphosphatase to thylakoid membranes

Aliphatic alcohols have a positive effect on the assoociation of pea ( Pisum sativum L. cv. Lincoln) chloroplast fructose- 1,6-bisphosphatase (FBPase; EC 3.1.3.11) with thylakoid membranes. The alcohol concentration needed to obtain a fixed percentage of enzyme association decreased with increased length of the aliphatic chain of the alcohol; maximum binding was obtained when the lysis medium contained, in molar fractions (or v/v percentages): 48×10^-4(T4 (2.4%), 26×10^-3 (10%), 40×10^-3 (15%), 76×10^-3 (21%), and 13×10^-2 (24%), of 1-butanol, 1-propanol, 2-propanol, ethanol, and methanol, respectively. A good correlation of binding with the octanol/water partition coefficient was observed. Since this coefficient constitutes a measure of hydrophobicity, we suggest that the binding of FBPase to the membranes occurs via hydrophobic clusters of both components.     

Inhibition of fructose 1,6-bisphosphatase by excess substrate and its reversal by monovalent cations

Purified rabbit liver fructose 1,6-bisphosphatase is maximally active with 2 μM fructose 1,6-bisphosphate. Above this concentration the substrate becomes inhibotory. Inhibition is reversed by $\text{NH}{}_4{}^{\mathrm{+}}$ or by physiological concentrations of K⁺. Substrate inhibition and its modification by monovalent cations may play a role in the regulation of gluconeogenesis at the step catalyzed by fructose 1,6-bisphosphatase.     

A novel nuclear protein in rat ventral prostate androgen-dependent and age-related change

A novel protein was found in the nuclei of rat ventral prostate. This protein has a molecular weight of about 21 kDa as measured by SDS-polyacrylamide gel electrophoresis. It showed a characteristic change between 3 and 84 weeks after birth in close association with the level of testosterone in the blood. After castration, the level of the 21-kDa protein decreased to $\text{1}\text{60}$ of normal in 7 days, but on daily injection of testosterone the level was restored to normal in 8 days and to twice the normal level in 14 days. Unlike H₁ and H₁⁰ histone and high mobility group proteins, the 21-kDa protein was not extracted with 5% HClO₄, but was partially extracted with 0.35 M NaCl. The 21-kDa protein was not found in kidney, liver, or brain, suggesting that it is specific to the ventral prostate.     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

Failure to corroborate claims that the developing endosperm of wheat (Triticum aestivum) contains significant activities of fructose-1,6-bisphosphatase

This work was done to test claims (Sangwan and Singh, Physiol. Plant. 73: 21–26) that the developing endosperm of wheat ( Triticum aestivum L.) contains a cytosolic and a plastidic fructose- 1,6-bisphosphatase (EC 3.1.3.11; FBPase). Repetition of the procedure of Sangwan and Singh with extracts of developing endosperm of Triticum aestivum cv. Mercia produced two peaks of apparent FBPase activity on elution from DEAE-cellulose. Both peaks showed high activity of pyrophosphate:fructose-6-phos-phate 1-phosphotransferase [EC 2.7.1.90; PFK(PP_i)]. The apparent FBPase activity in both peaks was stimulated by 20 μ M fructose-2,6-bisphosphate and inhibited by antibodies to PFK(PP_i). Antibody to plastidic FBPase did not react positively in an immunoblot analysis with any protein of M_r comparable to that of known FBPase in either peak. It is argued that the ability of each peak to convert fructose-1,6-bisphosphate to fructose-6-phosphate was due to PFK(PP_i). and that there remains no substantiated evidence for the presence of a plastidic FBPase in the developing endosperm of wheat.