Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.     

Presence of fructokinase in pancreatic islets

Homogenates of rat pancreatic islets that had been heated for 5 min at 70 degrees C to inactive hexokinases, catalyzed the ATP-dependent phosphorylation of D-fructose. This reaction was dependent on the presence of K+ and was inhibited by D-tagatose although not by D-glucose or D-glucose 6-phosphate. The phosphorylation product was identified as fructose 1-phosphate through its conversion to a bisphosphate ester by Clostridium difficile fructose 1-phosphate kinase. These findings allowed the conclusion that fructokinase (ketohexokinase) was responsible for this process. Similar results were observed with tumoral insulin-producing cells (RINm5F line). Fructokinase may account for a large share of fructose phosphorylation in intact islets, particularly in the presence of D-glucose.     

Estimation of fructokinase in crude tissue preparations

An assay for fructokinase activity is described that permits an accurate estimation of specific fructokinase in crude tissue preparations without interference of hexokinase activity. It utilizes two properties of hexokinases which differentiate hexokinase from fructokinase: (1) hexokinase activity is more labile to [H⁺] than is fructokinase, and (2) hexokinase activity is markedly inhibited by N-acetylglucosamine while fructokinase activity is relatively unaffected.     

Quantitative telomeric overhang determination using a double-strand specific nuclease

Telomeres terminate in 3′ overhangs that function in end protection and the formation of t-loops. Determining the steps and factors involved in overhang processing is compromised by the inability to easily and accurately determine overhang size in the presence of many kilobases of double-stranded telomeric DNA. We here describe the use of a double-strand specific nuclease (DSN) that entirely digests double-stranded DNA including telomeres, leaving the overhangs intact so that they can be measured.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.     

Expression of fructokinase isoforms in the sugarcane culm

Two isoforms of fructokinase (FRK), FRK1 and FRK2, are present in sugarcane (Saccharum spp. var N19) internodal tissue. Both isoforms are highly specific for fructose as the hexose substrate. FRK2 is inhibited by fructose concentrations exceeding 0.1 mM and 50% inhibition is attained at 230 μM (K_i (Fru) = 160 μM), while FRK1 activity is not negatively affected even at 1.0 mM fructose. The ratio of FRK2 to FRK1 activity is dependent on the developmental stage of the tissue. FRK1 appears to be the isoform that is preferentially expressed in mature tissue. Total FRK activity decreases during tissue maturation. This is the result of changes in expression of the isoforms and not inactivation of existing protein. A mathematical method that allows accurate estimation of the activities of the two isoforms of FRK in crude sugarcane extracts is presented.     

Bovine liver fructokinase: purification and kinetic properties.

Fructokinase from beef liver has been purified 2300-fold by acid and heat treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-100, DEAE- and CM-cellulose. The purified enzyme is homogeneous by all criteria examined, has a molecular weight of 56 000, and is a dimer of equal molecular weight subunits. The isoelectric point is 5.7. The Michaelis constant for activation by K+ is 15 mM, and the enzyme is also activated by Na+, Rb+, Cs+, NH4+, and TL+. The kinetic mechanism has been determined at pH 7.0, 25 degrees C. The initial velocity, product, and dead-end inhibition patterns for CrATP, CrADP, and 1-deoxy-D-fructose are consistent with a random kinetic mechanism with the formation of two dead-end complexes. Substrates for fructokinase include: D-fructose, L-sorbose, D-tagatose, D-psicose, D-xylulose, L-ribulose, D-sedoheptulose, L-galactoheptulose, D-mannoheptulose, 5-keto-D-fructose, D-ribose, 2,5-anhydro-D-mannitol, 2,5-anhydro-D-glucitol, 2,5-anhydro-D-mannose, 2,5-anhydro-D-lyxito.l, and D-ribono-gamma-lactone. 5-Thio-D-fructose was not a substate, but was a competitive inhibitor vs. D-fructose. Thus the minimum molecular for substrate activity seems to be (2R)-2-hydroxy-methyl-3,4-dihydroxytetrahydrofuran. The configuration of the substituents at carbons 3, 4, and 5 appears not to be critical, but the hydroxymethyl group must have the configuration corresponding to beta-D-(or alpha-L-) keto sugars. The anomeric hydroxyl on carbon 2 is not required (although it contributes to binding), and a wide variety of groups may be present at carbon 5.     

Carbon Partitioning in a Flaveria linearis Mutant with Reduced Cytosolic Fructose Bisphosphatase

Oxygen sensitivity and partitioning of carbon was measured in a mutant line of Flaveria linearis that lacks most of the cytosolic fructose-1,6-bisphosphatase found in wild-type lines. Photosynthesis of leaves of the mutant line was nearly insensitive to O₂, as found before. The mutant plants partitioned 2.5 times less carbon into sucrose than the wild type in a pulse chase experiment, with the extra carbon going mainly to starch but also to amino acids. From 10 to 50 min postlabeling, radioactivity chased out of the amino acid fraction to starch in both lines. In the middle of the light period, starch grains were larger in the mutant than in the wild type and covered 30% of the chloroplast area as seen with an electron microscope. Starch grains were found in both mesophyll and bundle sheath chloroplasts in both lines in these C₃-C₄ intermediate plants. At the end of the dark period, the starch levels were considerably reduced from what they were in the middle of the light in both lines. The concentration of sucrose was higher in the mutant line despite the lack of cytosolic fructose-1,6-bisphosphatase. The amino acid fraction accounted for about 30% of all label following a 10-min chase period. In the mutant line, most of the label was in the glycine + serine fraction, with 10% in the alanine fraction. In wild-type leaves, 35% of the label in amino acids was in alanine. These results indicate that this mutant survives the reduced cytosolic fructose-1,6-bisphosphatase activity by partitioning more carbon to starch and less to sucrose during the day and remobilizing the excess starch at night. However, these results raise two other questions about this mutant. First, why is the sucrose concentration high in a plant that partitions less carbon to sucrose, and second, why is alanine heavily labeled in the wild-type plants but not in the mutant plants?