Adipose-tissue phosphofructokinase. Rapid purification and regulation by phosphorylation in vitro.

A new procedure for the purification of phosphofructokinase using Blue Dextran-Sepharose is described. This allowed an approx. 1000-fold purification of phosphofructokinase from rat white and brown adipose tissue to be achieved in essentially a single step. The purified enzymes from both tissues were found to exhibit hyperbolic kinetics with fructose 6-phosphate, to be inhibited by ATP and citrate, and to be activated by 5'-AMP, phosphate and fructose 2,6-bisphosphate. The enzymes were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, and phosphorylation was found to be associated with increases in activity when the enzymes were assayed under appropriate sub-optimal conditions. In particular, the phosphorylated enzymes exhibited less inhibition by ATP and the white-adipose-tissue enzyme was more sensitive to activation by fructose 2,6-bisphosphate. It is suggested that an increase in the cytoplasmic concentration of cyclic AMP in tissues other than liver may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.     

Phosphofructokinase from mollusc muscle is activated by phosphorylation.

Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.     

Inhibition of alpha-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase.

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na+-dependent alpha-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport. The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intravesicular contents and loosely associated membrane proteins. 32P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the 32P from [gamma-32P]ATP is incorporated into protein phosphomonoester linkages.     

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes.

The addition of glucagon (10^?6 M) to an incubation mixture containing ³²Pi and hepatocytes isolated from livers of rats fed $\text{ad libitum}$ results in both a 3-fold increased incorporation of ³²P into L-type pyruvate kinase and a decreased catalytic activity. The ³²P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10^?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10^?7 M) had essentially no effect on the incorporation of ³²P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase $\text{invivo}$ is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythrocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin) kinase according to physical and site-specificity criteria. The soluble enzyme shows an Mr of about 30000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that had been phosphorylated with [gamma-32P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with 32Pi. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid

Two-dimensional, high-resolution electrophoretic technique of O'Farrell has been adapted to the analysis of thyroid phosphorylated proteins. Proteins were extracted from dog thyroid slices which had been incubated in the presence of [32P]phosphate with thyrotropin or with different agents which enhance the intracellular accumulation of cyclic AMP. About 350 phosphorylated polypeptides have been separated. Thyrotropin stimulates the phosphorylation of at least eight of these polypeptides. An increase in the phosphorylation of the same polypeptides was observed was observed when dog thyroid slices were incubated with dibutyryl adenosine 3':5'-monophosphate, cholera toxin or prostaglandin E1 instead of thyrotropin. Our results confirm that most of dog thyroid protein phosphorylation is independent of cyclic AMP. They offer a first link between the action of cyclic AMP on protein kinase and the physiological effects of thyrotropin. They strongly substantiate the hypothesis that most thyrotropin effects are mediated by cyclic AMP.     

Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver

Low phosphate and high phosphate forms of phosphofructokinase (Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255, 11656-11659) from rat liver were purified to homogeneity and various properties were compared. The specific activities of these enzymes and their electrophoretic mobilities on polyacrylamide in sodium dodecyl sulfate are the same. A limited tryptic digestion yields products with no change in the enzyme activity but with a reduction in the molecular weight of about 2000. Both low and high phosphate enzymes can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, and approximately twice as much [32P]phosphate is incorporated into the low phosphate than the high phosphate enzyme. A comparison of their allosteric kinetic properties reveal that the high phosphate enzyme is much more sensitive to inhibition by ATP and citrate and shows a higher K0.5 for fructose 6-phosphate than the low phosphate enzyme, and the difference in the K0.5 values becomes greater at lower pH values. Furthermore, the high phosphate phosphofructokinase is less sensitive to activation by AMP and fructose 2,6-bisphosphate. Moreover, when the low phosphate enzyme is phosphorylated by protein kinase, the resulting phosphorylated enzyme exhibits a higher K0.5 for fructose 2,6-bisphosphate than does the untreated enzyme. These results demonstrate that the phosphorylation affects the allosteric kinetic properties of the enzyme and results in a less active form of phosphofructokinase.     

Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum.

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Adenosine 3′,5′-monophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum

In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fraction by cyclic AMP and luteinizing hormone was reported (Menon, K.M.J. (1973) J. Biol. Chem. 248, 494–501). We have now studied the effects of cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteins. Extraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis. The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormone. At least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the case of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP whereas luteinizing hormone has no effect on ribosome phosphorylation.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythorocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin_kinase according and site-specificity criteria. The soluble enzyme shows an M_r of about 30 000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32 000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that has been phosphorylated with [γ-³²P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21 500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with ³²P_i. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Role of dehydration in changing the permeability of erythrocyte plasma membranes by freeze-thawing

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing 32Pi, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weights between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [gamma-32P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [gamma-32P]ATP and cyclic AMP-dependent protein kinase from calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Inhibition of α-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na⁺-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport.The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. ³²P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the ³²P from [γ-³²P]ATP is incorporated into protein phosphomonoester linkages.     

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.     

Engineering of carbon distribution between glycolysis and sugar nucleotide biosynthesis in Lactococcus lactis

We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an approximately 100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.