Enzymatic properties of pyruvate kinase from the rumen ciliates genus Entodinium.

1. Pyruvate kinase from the rumen ciliates genus Entodinium was partially purified and the enzymatic properties were investigated. 2. Three types of pyruvate kinase (type I, II and III) on DEAE-cellulose column were eluted with a linear gradient of KCl. The enzymatic properties differed among the types of enzyme, especially type I and type III displayed different kinetic properties to each other. The enzymatic property of type II enzyme was an intermediate between type I and III. 3. The principal enzyme, type I, required a divalent cation, Mg2+ and was activated with AMP and FDP. ATP was a potent inhibitor. The saturation curves for the substrates, PEP and ADP, were hyperbolic and Km values were 0.15 and 0.27 mM, respectively.

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts.

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.

ACTH stimulates fructose 2,6-bisphosphate synthesis and glycolysis in Y-1 adrenal tumor cells.

The effect of ACTH on glycolysis has been studied in Y-1 tumor adrenal cells. ACTH caused a sustained increase in the liberation of lactate as well as a stimulation of both basal and glucose-induced fructose 2,6-bisphosphate content. ACTH produces changes also in the activities of phosphofructokinase-1 and phosphofructokinase-2. The addition of Ca2+ or dibutyryl cyclic AMP did not modify neither lactate production nor fructose 2,6-bisphosphate levels. The results suggest that fructose 2,6-bisphosphate regulates ACTH-induced glycolysis at the phosphofructokinase-1 step, although the biochemical mechanism of phosphofructokinase-2 activation remains elusive.

Phosphofructokinase in rat lung during perinatal development: characterization of subunit composition and regulation by fructose 2,6-bisphosphate and glucose 1,6-bisphosphate.

The subunit composition of phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) was studied in rat lung during perinatal development. No change in subunit composition during this period was observed. The three subunits of phosphofructokinase (L, M and C) were present in a ratio of approx. 65:25:10, respectively. In addition the levels of two effectors of phosphofructokinase were determined in rat lung during perinatal development: glucose 1,6-bisphosphate and fructose 2,6-bisphosphate. Until day 20 of gestation (term is 22 days) the glucose 1,6-bisphosphate level remains relatively constant (approx. 0.55 mumol/g protein), decreases before birth and increases sharply up to 1.04 mumol/g protein 2 days after birth. The amount of fructose 2,6-bisphosphate in rat lung shows a different developmental profile. A small peak is shown at day 17 of gestation whereas a larger peak up to 36.4 nmol/g protein is shown at days 20 and 21 of gestation. The time of maximal fructose 2,6-bisphosphate content corresponds with the time of glycogen breakdown and acceleration of surfactant synthesis in prenatal rat lung. Both glucose 1,6-bisphosphate and fructose 2,6-bisphosphate stimulate lung phosphofructokinase. Half maximal stimulations occur in the range of 24.1-70.9 microM glucose 1,6-bisphosphate and 0.17-0.34 microM fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate in Dictyostelium discoideum. Independence of cyclic AMP production and inhibition of fructose-1,6-bisphosphatase.

The occurrence of fructose 2,6-bisphosphate was detected in Dictyostelium discoideum. The levels of this compound were compared with those of cyclic AMP and several glycolytic intermediates during the early stages of development. Removal of the growth medium and resuspension of the organism in the differentiation medium decreased the content of fructose 2,6-bisphosphate to about 20% within 1 h, remaining low when starvation-induced development was followed for 8 h. The content of cyclic AMP exhibited a transient increase that did not correlate with the change in fructose 2,6-bisphosphate. If after 1 h of development 2% glucose was added to the differentiation medium, fructose 2,6-bisphosphate rapidly rose to similar levels to those found in the vegetative state, while the increase in cyclic AMP was prevented. The contents of hexose 6-phosphates, fructose 1,6-bisphosphate and triose phosphates changed in a way that was parallel to that of fructose 2,6-bisphosphate, and addition of sugar resulted in a large increase in the levels of these metabolites. The content of fructose 2,6-bisphosphate was not significantly modified by the addition of the 8-bromo or dibutyryl derivatives of cyclic AMP to the differentiation medium. These results provide evidence that the changes in fructose 2,6-bisphosphate levels in D. discoideum development are not related to a cyclic-AMP-dependent mechanism but to the availability of substrate. Fructose 2,6-bisphosphate was found to inhibit fructose-1,6-bisphosphatase activity of this organism at nanomolar concentrations, while it does not affect the activity of phosphofructokinase in the micromolar range. The possible physiological implications of these phenomena are discussed.

Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms.

The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.

Interference by ATPase in the assay of rat heart phosphofructokinase.

A high molecular-weight protein was found in heart extracts which, in the assay for phosphofructokinase, artificially activated the enzyme. The protein could be removed by gel-exclusion chromatography or high-speed centrifugation. The mechanism of activation appeared to be due to the hydrolysis of ATP to ADP, AMP and inorganic phosphate which was inhibited by Mn2+. Phosphofructokinase is thus activated by the production of activators and by the lowered inhibitory concentration of ATP. Since the adrenergic/Ca2+-activated form of the enzyme is the more sensitive to activators, the difference between the two forms of phosphofructokinase is amplified in the presence of the ATPase and diminished upon its removal or its inhibition by Mn2+. The Mn2+-sensitive ATPase appears to play no part in the adrenergic/Ca2+-mediated control of cardiac phosphofructokinase or the interconverting reactions.

Regulation of glucose metabolism in rat adrenal gland in alloxan-diabetes: the possible role of fructose 2,6-bisphosphate.

The level of fructose 2,6-bisphosphate is markedly decreased in the rat V.renal gland in diabetes, falling to 23% of the control value. There is parallel decrease in the flux of 14C-labelled glucose through the glycolytic route and tricarboxylic acid cycle. Only minimal changes in hexokinase (EC 2.7.1.1.), a 22% decrease in Type I hexokinase of the soluble fraction, were observed, highlighting the probable significant involvement of fructose 2,6-bisphosphate in the regulation of glycolysis in the adrenal. In contrast, there was evidence for a marked rise in the flux of glucose through the pentose phosphate pathway, which may be linked to enhanced corticoid synthesis in the diabetic state.

Role of fructose 2,6-bisphosphate in the regulation of glycolysis in various types of cultivated brain cell.

The stimulation of glycolysis by glucose and anoxia has been investigated in cultivated rat cerebellar neurons, mice neuroblastoma cells and rat brain astrocytes. It is shown that fructose 2,6-bisphosphate exerts no predominant role in: (1) the stimulation of glycolysis by glucose, and (2) the onset of anaerobic glycolysis.

Pyruvate kinase isozyme (PK-Greenville) with defective allosteric activation by fructose-1,6-diphosphate: the role of F-1,6-P modulation in normal erythrocyte metabolism.

A child with chronic hemolytic anemia since birth was found to have erythrocyte pyruvate kinase (PK) in a highly unusual form relative to other mutant isozymes when characterized by International Committee for Standardization in Hematology criteria. Most properties of the partially purified isozyme (designated PK-Greenville) were altered minimally, if at all, except for nearly total insensitivity to allosteric activation by fructose-1,6-diphosphate (F-1,6-P). One parent appeared to be heterozygous for a null gene and the other for an allele governing production of the mutant isozyme. Apparent restriction of the molecular defect to ineffective activation kinetics suggests that the F-1,6-P binding site on erythrocyte PK is functionally as well as physically allosteric. The magnitude of the metabolic block at the PK step and the clinical severity indicate that allosteric modulation by F-1,6-P is a crucial property of PK in normal erythrocyte metabolism.

A fructose bisphosphate activated lactate dehydrogenase in the liver fluke Fasciola hepatica.

Lactate dehydrogenase of Fasciola hepatica showed typical Michaelis-Menten kinetics at pH 7.2, with respect to pyruvate. Addition of physiological levels of fructose bisphosphate activated the enzyme at all substrate concentrations tested; the response to this effector being hyperbolic in nature. As well as depending upon the fructose bisphosphate concentration, the Vmax and Km are modified by different buffers. The degree of activation is much greater using Tris-HCl than phosphate buffer. The pH optimum occurs at pH 6.5 whether using physiological levels of substrate in the presence or absence of fructose bisphosphate, or high levels of substrate. Of the potential effectors tested, significant inhibition was shown by the nucleoside triphosphates, especially ATP. The importance of this inhibition, coupled with the activation by fructose bisphosphate is discussed. Fasciola hepatica lactate dehydrogenase is unusual in that it does not catalyse the reverse reaction to any measurable extent. That is, lactate oxidation is negligible unless the effector fructose bisphosphate is present. Use was made of this fact to visualise the isoenzymes of lactate dehydrogenase separated by polyacrylamide disc gel electrophoresis. Five isoenzyme bands became apparent when stained in this manner.

Fructose-2,6-P2, chemistry and biological function.

A new activator of phosphofructokinase, which is bound to the enzyme and released during its purification, has been discovered. Its structure has been determined as beta-D Fructose-2,6-P2 by chemical synthesis, analysis of various degradation products and NMR. D-Fructose-2,6-P2 is the most potent activator of phosphofructokinase and relieves inhibition of the enzyme by ATP and citrate. It lowers the Km for fructose-6-P from 6 mM to 0.1 mM. Fructose-6-P,2-kinase catalyzes the synthesis of fructose-2,6-P2 from fructose-6-P and ATP, and the enzyme has been partially purified. The degradation of fructose-2,6-P2 is catalyzed by fructose-2,6-bisphosphatase. Thus a metabolic cycle could occur between fructose-6-P and fructose-2,6-P2, which are catalyzed by these two opposing enzymes. The activities of these enzymes can be controlled by phosphorylation. Fructose-6-P,2-kinase is inactivated by phosphorylation catalyzed by either cAMP dependent protein kinase or phosphorylase kinase. The inactive, phospho-fructose-6,P,2-kinase is activated by dephosphorylation catalyzed by phosphorylase phosphatase. On the other hand, fructose-2,6-bisphosphatase is activated by phosphorylation catalyzed by cAMP dependent protein kinase. Investigation into the hormonal regulation of phosphofructokinase reveals that glucagon stimulates phosphorylation of phosphofructokinase which results in decreased affinity for fructose-2,6-P2 appears to be due to the decreased synthesis by inactivation of fructose-2,6-P2,2-kinase and increased degradation as a result of activation of fructose-2,6-bisphosphatase. Such a reciprocal change in these two enzymes has been demonstrated in the hepatocytes treated by glucagon and epinephrine. The implications of these observations in respect to possible coordinated controls of glycolysis and glycogen metabolism are discussed.