Studies on the Formation and the Degradation of Glucose 1,6-Bisphosphate in the Beef Liver Homogenate

Four kinds of the enzyme reactions have been reported for the synthesis of Glc-1,6-P₂. However, any activity of Glc-1-P dismutase and phosphoglucokinase was not observed in the beef liver homogenate. When the liver homogenate was incubated with Glc-1-P and Fru-1,6-P₂, a significant amount of Glc-1,6-P₂ was formed. The Glc-1,6-P₂ synthesis activity from Glc-1-P and Fru-1,6-P₂ was caused by the action of phosphoglucomutase present in the liver homogenate. The most remarkable activity for Glc-1,6-P₂ synthesis was observed when the homogenate was incubated with Glc-1-P and glycerate-1,3-P₂. The Glc-1,6-P₂ synthesis activity from Glc-1-P and glycerate-1,3-P₂ was separated from the major peak of phosphoglucomutase activity by DEAE-Sephadex chromatography. The peak of Glc-1,6-P₂ synthesis activity, however, still retained phosphoglucomutase activity.

Glc-1,6-P₂ phosphatase activity was mainly observed in the mitochondria and microsome fraction. The properties of Glc-1,6-P₂ phosphatase were differentiated from those of acid phosphatase and Glc-6-P phosphatase.     

Purification and Properties of Beef Liver Phosphoglucomutase

A procedure is described for purification of phosphoglucomutase [EC 2.7.5.1] from beef liver. The purified enzyme preparation was homogeneous on the analysis of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be 64,000 by the meniscus depletion method.

The amino acid composition of liver phosphoglucomutase was very similar to that of the rabbit muscle enzyme.

The reaction mechanism of liver phosphoglucomutase was examined kinetically. The results of kinetical experiments strongly suggested that the reaction of liver phosphoglucomutase proceeds via “ping-pong” mechanism.

Liver phosphoglucomutase activity was remarkably inhibited by Fru-1,6-P₂, glycerate-2,3-P₂ and glycerate-1,3-P₂. Role of the bisphosphate compounds on the control of carbohydrate metabolism is discussed.     

The purification and kinetic properties of biophosphoglycerate synthase from horse red blood cells.

Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P₂ or glycerate-2,3-P₂ to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P₂ → glycerate-2,3-P₂), k_cat 12.5 s^?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P₂ → glycerate-P + P_i), k_cat 2.6 s^?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), k_cat 1.7 s^?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell.     

A specific enzyme for glucose 1,6-bisphosphate synthesis.

The reaction: glycerate-1,3-P2 PLUS GLUCOSE-1-P YIELDS TO GLUCOSE-1,6-P2 plus glycerate-P is catalyzed by a distinct enzyme of mouse brain. A divalent metal requirement was shown when the enzyme was treated with imidazole and EDTA. Mg2+, Mn2+, Ca2+, Zn2+, Ni2+, Co2+, and Cd2+ were quite effective cofactors. The enzyme, in better than 50 percent yield, has been purified away from 99 percent of the phosphoglucomutase, phosphoglycrate mutase, and phosphofructokinase. Acetyl-P, ATP, enolpyruvate-P, creatine-P, and fructose-1,6-P2 are not phosphoryl donors. Glucose-6-P and mannose-1-P are good alternate acceptors. Mannose-6-P, galactose-Ps, and fructose-Ps have little or no acceptor activity. Strong inhibition was found with fructose-1,6-P2, glycerate-2,3-P2, enolpyruvate-P, and acetyl CoA. From the amount of activity and the kinetic constants of the purified enzyme it seems likely that this enzyme is responsible for the glucose-1,6-P2 synthesis of brain.     

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

Mammalian phosphomannomutase PMM1 is the brain IMP-sensitive glucose-1,6-bisphosphatase

Glucose 1,6-bisphosphate (Glc-1,6-P(2)) concentration in brain is much higher than what is required for the functioning of phosphoglucomutase, suggesting that this compound has a role other than as a cofactor of phosphomutases. In cell-free systems, Glc-1,6-P(2) is formed from 1,3-bisphosphoglycerate and Glc-6-P by two related enzymes: PGM2L1 (phosphoglucomutase 2-like 1) and, to a lesser extent, PGM2 (phosphoglucomutase 2). It is hydrolyzed by the IMP-stimulated brain Glc-1,6-bisphosphatase of still unknown identity. Our aim was to test whether Glc-1,6-bisphosphatase corresponds to the phosphomannomutase PMM1, an enzyme of mysterious physiological function sharing several properties with Glc-1,6-bisphosphatase. We show that IMP, but not other nucleotides, stimulated by >100-fold (K(a) approximately 20 mum) the intrinsic Glc-1,6-bisphosphatase activity of recombinant PMM1 while inhibiting its phosphoglucomutase activity. No such effects were observed with PMM2, an enzyme paralogous to PMM1 that physiologically acts as a phosphomannomutase in mammals. Transfection of HEK293T cells with PGM2L1, but not the related enzyme PGM2, caused an approximately 20-fold increase in the concentration of Glc-1,6-P(2). Transfection with PMM1 caused a profound decrease (>5-fold) in Glc-1,6-P(2) in cells that were or were not cotransfected with PGM2L1. Furthermore, the concentration of Glc-1,6-P(2) in wild-type mouse brain decreased with time after ischemia, whereas it did not change in PMM1-deficient mouse brain. Taken together, these data show that PMM1 corresponds to the IMP-stimulated Glc-1,6-bisphosphatase and that this enzyme is responsible for the degradation of Glc-1,6-P(2) in brain. In addition, the role of PGM2L1 as the enzyme responsible for the synthesis of the elevated concentrations of Glc-1,6-P(2) in brain is established.     

Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle.

The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.     

Effect of vanadate on the glucose-1,6-bisphosphate synthase and glucose-1,6-bisphosphatase activities of phosphoglucomutase

Phosphoglucomutase, in addition to catalyzing the interconversion of glucose 1-P and glucose 6-P, catalyzes both the synthesis of glucose 1,6-P2 from glucose monophosphate and either fructose 1,6-P2 or glycerate 1,3-P2, and the hydrolysis of glucose 1,6-P2. Vanadate inhibits the mutase activity, activates the synthase activities, and does not affect the phosphatase activity. These effects suggest that the "exchange" step postulated for the phosphoglucomutase pathway is specifically inhibited by vanadate.     

On the biochemical basis of hereditary fructose intolerance.

In hereditary fructose intolerance it was found that in addition to an increased K_m value for Fru-1-P, the K_m of aldolase for Fru-1,6-P₂ was also increased. Furthermore, human phosphorylase $\text{a}$ was found to be inhibited by Fru-1-P in a non-competitive way.     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria

A production system of UDP-N-acetylglucosamine (UDP-GlcNAc) was established by using recombinant Escherichia coli and Corynebacterium ammoniagenes in combination. E. coli overexpressed the UDP-GlcNAc biosynthetic genes, glmM, glmU, glk, ppa, ack, and pta, whereas C. ammoniagenes contributed to the formation of UTP from orotic acid. Glucose 1,6-diphosphate (Glc-1,6-P₂), which was required for the activity of phosphoglucosamine mutase involved in UDP-GlcNAc biosynthesis, was supplied by phosphoglucomutase and phosphofructokinase. Starting with orotic acid (65 mM) and glucosamine (400 mM), UDP-GlcNAc accumulated at 11.4 mM (7.4 g l^–1) after 8 h.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

Metabolism of glycerate-2,3-P2--II. Enzymes involved in the glycerate-2,3-P2 metabolism in chicken skeletal muscle

1. Four enzyme fractions which may be involved in the synthesis and breakdown of glycerate-2,3-P2 have been isolated from extracted skeletal muscle by gel-filtration and ion-exchange chromatography. 2. One of the fractions, corresponding to the glycerate-2,3-P2 dependent phosphoglycerate mutase, has been purified to homogeneity. In addition to the main enzymatic activity, it shows intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity stimulable by glycolate-2-P. Its synthase activity represents about 10% of the total synthase activity of the tissue, and its phosphatase activity corresponds to about 60% of the total phosphatase activity. 3. Two of the fractions have glycerate-2,3-P2 synthase, glycerate-2,3-P2 phosphatase and phosphoglycerate mutase activities in a ratio similar to that of the glycerate-2,3-P2 synthase described in mammalian skeletal muscle. Their synthase activity corresponds to about 90% of the total synthase activity, and their phosphatase activity represents about 1% of the total phosphatase activity of the tissue. 4. The fourth fraction shows only glycerate-2,3-P2 phosphatase activity and represents about 40% of the total activity of the tissue. 5. It is suggested that in chicken skeletal muscle the metabolism of the glycerate-2,3-P2 is regulated in a way similar to that described in mammalian skeletal muscle.     

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

Summary A quantative cytochemical assay for PP_i-PFK activity in the presence of Fru-2,6-P₂ is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PP_i-PFK activity, has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PP_i-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.Abbreviations AMP adenosine monophosphate - ATP adenosine triphosphate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Fru-2,6-P₂ fructose 2,6-bisphosphate - Fru-6-P fructose 6-phosphate - FB Pase 2 fructose 2,6-bisphosphatase (EC 3.1.3.46) - Gl-3-PD glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide - NBT nitroblue tetrazolium - PEP phosphoenolpyruvate - PFK 6-phosphofructokinase (EC 2.7.1.11) - PFK₂ 6-phosphofructo-2-kinase (EC 2.7.1.105) - PP_i pyrophosphate - PP_i-PFK pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) - PVA polyvinyl alcohol (G04/140 Wacke Chemical Company)     

The participation of glucose-1,6-diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats

1. The level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was found to be strikingly decreased in brains of adult rats (5 months of age) as compared to young (10-14 days of age). 2. This age-related decrease in Glc-1,6-P2, the potent inhibitor of hexokinase and activator of phosphoglucomutase, was accompanied by a correlated increase in the activity of hexokinase and a reduction in phosphoglucomutase. 3. Evidence is provided showing that Glc-1,6-P2 participates in the regulation of these enzymes' activities with age. 4. The age-related changes in Glc-1,6-P2 and in the enzymes' activities in brain were opposite to those which we previously found in skeletal muscle. 5. These results suggest that Glc-1,6-P2 is involved in the regulation of carbohydrate metabolism during growth in both brain and muscle, as well as in the interrelationship between these two tissues.     

Altered glucose 1,6-bisphosphate and fructose 2,6-biphosphate levels in low-frequency stimulated rabbit fast-twitch muscle

Glucose 1,6-bisphosphate (Glc-1,6-P2) and fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations display pronounced increases in rabbit fast-twitch muscle during chronic low-frequency stimulation. These increases are first seen after stimulation periods exceeding 3 h and reach maxima after 12-24 h of stimulation (approximately 3-fold for Glc-1,6-P2 and 5-fold for Fru-2,6-P2). Both metabolites regress to normal values after stimulation periods longer than 4 days. The fact that their increases coincide with the replenishment of glycogen after its initial depletion, could point to a role of Glc-1,6-P2 and Fru-2,6-P2 in glycogen metabolism.     

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.     

Increase in glucose 1,6-bisphosphate levels, activation of phosphofructokinase and phosphoglucomutase, and inhibition of glucose 1,6-bisphosphatase in muscle induced by trifluoperazine

Injection of trifluoperazine (TFP) to rats induced a significant rise in the level of glucose 1,6-bisphosphate (Glc-1,6-P2) in muscle. This increase in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked activation of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. Glucose-1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2) was markedly inhibited following the injection of TFP, which may account for the rise in the Glc-1,6-P2 level. Previous results from this laboratory have revealed that muscle damage or weakness is characterized by a decrease in Glc-1,6-P2 levels, leading to a marked reduction in the activities of phosphoglucomutase and phosphofructokinase (the rate-limiting enzyme in glycolysis). The present results suggest that TFP treatment may have a beneficial effect on the depressed glycolysis in muscle weakness or damage.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

Studies on the entry of fructose-2,6-bisphosphate into chloroplasts

The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P₂) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P₂ controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate, fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P₂ is present mainly in the cytosol. Here we present evidence that Fru-2,6-P₂ can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P₂ (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P₂ in the stroma. Elevated plastid Fru-2,6-P₂ levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P₂ enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6-P₂ on chloroplast enzymes.