Prime Ricerche sulla Fisionomia Enzimatica Caratteristica della Fotosintesi

Summary

The rate of oxidation of glucose-6-phosphate, ribose-5-phosphate, fructose-1,6- phosphate, iso-citrate and malate in extracts from green and etiolated pea leaves was determined, using the triphenyltetrazolium technique.

Glucose-6-phosphate, ribose-5-phosphate, and fructose-1,6-phosphate, in the presence of added TPN, where oxidized at a rate about twice higher in the extracts from green than in the extracts from etiolated leaves. Iso-citrate, in the presence of TPN, fructose-1,6-phosphate, in the presence of DPN, and malate, in the presence of either TPN or DPN, were oxidized at about the same rate in the two types of extracts.

These data seem to indicate a preferential synthesis of enzymes involved in the metabolic cycle of phosphorylated sugars during the transition of the leaf from the etiolated to the photosynthetising physiognomy. They seem also favourable to the view assigning to this metabolic system a primary importance in the anabolic pathway of photosynthesis.     

Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12

Summary We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.Abbreviations G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1-6DP fructose-1,6-diphosphate - PEP phosphoenol pyruvate     

Inhibition of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in sea urchin eggs by palmitoyl-coenzyme a and reversal by polyamines

Palmitoyl-CoA inhibited crude glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the eggs of the sea urchin, Hemicentrotus pulcherrimus. Fifty percent inhibition of the glucose 6-phosphate dehydrogenase in the supernatant of unfertilized eggs was obtained with 0.43 ± 0.05 μm palmitoyl-CoA, and of 6-phosphogluconate dehydrogenase with 4.41 ± 0.20 μm palmitoyl-CoA. Also, these enzymes in fertilized eggs 30 min after fertilization were inhibited by palmitoyl-CoA almost as much as in unfertilized eggs. Na-Palmitate, coenzyme A, acetyl-CoA, palmitoylcarnitine, and carnitine failed to exert any inhibitory effect on the activities of these dehydrogenases. The intracellular concentration of long-chain fatty acyl-CoA in unfertilized eggs (3.08 ± 0.33 nmol/10⁶ eggs) was high enough for the inhibition of these enzymes, and decreased following fertilization to a low level (1.49 ± 0.08 nmol/10⁶ eggs 30 min after fertilization). Spermine and spermidine canceled the inhibition of these enzymes by palmitoyl-CoA. In view of the inhibition of glucose 6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase by palmitoyl-CoA, these dehydrogenases in the pentose monophosphate cycle are probably inhibited in unfertilized eggs by long-chain fatty acyl-CoA and released from the inhibited state by both the decrease in the level of long-chain acyl-CoA and the increase in the level of polyamines following fertilization.     

Do non-equilibrium reactions in the glucose-to-triose phosphate pathway exist in the liver?

The circadian changes in the contents of intermediates of the initial reactions of the glycolytic pathway in pigeon liver were studied. the concentrations of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate and triose phosphates were found to change synchronously, being maximal at the dark time and minimal during the light daytime. The glycogen content in the liver decreased steadily between 12.00 and 09.00. The diurnal variations in the concentrations of metabolite pairs (glucose and glucose-6-phosphate, glucose-6-phosphate and fructose-6-phosphate, fructose-6-phosphate and fructose-1.6-diphosphate, fructose-1.6-diphosphate and triose phosphates) appeared to correlate significantly. The results obtained suggest that in the liver at least there are no limiting i. e. physiologically non-equilibrium reactions in the carbohydrate metabolic pathway from glucose to triose phosphates.     

The change in osmotically inactive fraction produced by cell activation

1. Resting and activated eggs of the sea urchin Arbacia punctulata were swollen in hypotonic sea water (60, 70, 80, and 90 per cent), and allowed to attain equilibrium volumes (Figs. 1 and 2). 2. Both fertilized and unfertilized eggs obey the Boyle-van't-Hoff law, but the value for b, the "osmotically inactive fraction" or non-swellable volume, was different for the two, averaging in the cases studied 7.3 per cent for unfertilized and 27.4 per cent for fertilized. 3. On activation, the eggs of the sea urchin undergo a definite increase in total cell volume, of approximately 2.7 per cent. 4. Some evidence is adduced for the possibility that the alteration in cell volume and in o.i.f. may depend upon the species in question. 5. A parallelism between change in b and alteration of respiratory metabolism in Arbacia, Chaetopterus, and Arbacia fragments is pointed out. This requires further investigation in other species to establish generality. 6. Equations for the calculation of the point at which osmotic pressures and cell volumes are identical for unfertilized and fertilized eggs are included. 7. A mechanical analogue of the phenomena is introduced (Fig. 3).     

Catalytic properties of phosphoglucomutase from pea chloroplasts.

Electrophoretically homogeneous phosphoglucomutase (PGM) with specific activity of 3.6 units/mg protein was isolated from pea (Pisum sativum L.) chloroplasts. The molecular mass of this PGM determined by gel-filtration is 125 +/- 4 kD. According to SDS-PAGE, the molecular mass of subunits is 65 +/- 3 kD. The Km for glucose-1-phosphate is 18.0 +/- 0.5 microM, and for glucose-1, 6-diphosphate it is 33 +/- 0.7 microM. At glucose-1-phosphate and glucose-1,6-diphosphate concentrations above 0.5 and 0.2 mM, respectively, substrate inhibition is observed. The enzyme has optimum activity at pH 7.9 and 35 degrees C. Mg2+ activates the PGM. Mn2+ activates the enzyme at concentrations below 0.2 mM, while higher concentrations have an inhibitory effect. The activity of the PGM is affected by 6-phosphogluconate, fructose-6-phosphate, NAD+, ATP, ADP, citrate, and isocitrate.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VII. OBSERVATIONS ON THE AMOUNT AND POSSIBLE FUNCTION OF DIPHOSPHOTHIAMINE (COCARBOXYLASE) IN EGGS OF ARBACIA PUNCTULATA

1. Methods suitable for the determination of diphosphothiamine (cocarboxylase) in eggs of Arbacia punctulata have been developed. Quantitative extraction of the cocarboxylase was effected by combining the use of thiamine hydrochloride in the extraction fluid with critical adjustment of the pH of extraction to pH 6.3–6.7. 2. The unfertilized eggs were found to contain the equivalent of 2 to 3 micrograms of natural yeast cocarboxylase per gm. of wet eggs; the cocarboxylase content of the 30 minute and 10 hour fertilized eggs was somewhat less (Table III). 3. In preliminary experiments, Arbacia egg cytolysates were found to cause pyruvic acid to disappear. The rate of such disappearance was apparently greater under aerobic than under anaerobic conditions; it was also greater for cytolysates from fertilized eggs than for cytolysates from unfertilized eggs (Table IV).     

Glucose-6-phosphate-dependent pyruvate kinase in Streptococcus mutans.

Pyruvate kinase of Streptococcus mutans JC 2 had an absolute and specific requirement for glucose-6-phosphate. Inorganic phosphate was a strong inhibitor. The enzyme required K+ or NH4+ and Mg2+ or Mn2+. S. mutans FIL and E 49, Streptococcus bovis ATCC 9809, and Streptococcus salivarius ATCC 13419 had also glucose-6-phosphate-dependent pyruvate kinases, whereas Streptococcus sanguis NCTC 10904 had an enzyme activated by fructose-1,6-diphosphate.     

Glucose-induced accumulation of glucose-1,6-bisphosphate in pancreatic islets : its possible role in the regulation of glycolysis

A rise in the extracellular concentration of glucose from zero to 5.6 and 16.7 mM caused a graded increase in the glucose-1,6-bisphosphate content of rat pancreatic islets. Glucose-1,6-bisphosphate activated phosphofructokinase in islet homogenates, when the reaction velocity was measured at low concentrations of fructose-6-phosphate. It is postulated that glucose-1, 6-bisphosphate participates, together with fructose-2,6-bisphosphate, in the regulation of glycolysis in intact islet cells.     

Mechanism for Regulating the Distribution of Glucose Carbon Between the Embden-Meyerhof and Hexose-Monophosphate Pathways in Streptococcus faecalis

Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.     

Inhibition of glucose in Zajdela hepatoma by 6-phosphogluconate; the role of 3-phosphoglycerate

Influence of 6-phosphogluconate and 3-phosphoglycerate have been studied for their effect on the fructose-6-phosphate glycolytic transformation reactions in homogenates of the Zajdela hepatoma cells. It is established that 6-phosphogluconate inhibits formation of lactate from fructose-6-phosphate and increases the ratio: dioxyacetone-phosphate/lactate. The influence of 6-phosphogluconate on the formation of lactate from the fructose-1,6-bisphosphate is similar. 3-phosphoglycerate removes the effect of 6-phosphogluconate, its content being unchanged in samples, which indicates rather the regulatory, than the substrate role of 3-phosphoglycerate. Analogous experiments with homogenates of the rat liver show that 6-phosphogluconate inhibits hexosephosphate isomerase, but almost all the introduced substrate (fructose-6-phosphate) is transformed into glucose. Processes of fructose-6-phosphate consumption in the hepatoma and liver are opposite.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.     

Distribution of some enzymes concerning carbohydrate metabolism in sea urchin eggs

As an aid to the elucidation of the mechanism of activation of glycolysis upon fertilization, the activity and the distribution of the enzymes concerned were measured in unfertilized and fertilized eggs of Hemicentrotus pulcherrimus and Pseudocentrotus depressus. The enzymes investigated were phosphorylase, exo-1,4-α-glucosidase, hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, 6-phosphofructokinase, hexosediphosphatase, fructose-bisphosphate aldolase, pyruvate kinase, and lactate dehydrogenase.Phosphorylase and pyruvate kinase were the enzymes which were activated upon fertilization. Glucose-6-phosphate dehydrogenase and a part of aldolase changed their distribution from the particulate to the soluble fraction upon fertilization. Advantages of enzyme activation over changes in enzyme distribution upon fertilization were discussed as a mechanism for the fertilization-induced activation of glycolysis.     

Oxidative Enzymes and Pathways of Hexose and Triose Metabolism in Chlorella

Cell-free preparations of Chlorella pyrenoidosa Chick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared. The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose-6-phosphate, 6-phosphogluconate, and fructose-1-6-diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose-diphosphate-aldolase, NAD-linked 3-phosphoglyceraldchyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and a NADP-linked 3-phosphoglyceraldchyde dehydrogenase. In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose-6-phosphate and 6-phosphogluconate in relation to the reduction of NAD by 3-phosphoglyceraldehyde was made in light- and dark-grown cells. The rate of reduction of NADP appeared to be lowered in the light-grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophic metabolism than in heterotrophic metabolism.     

Purification and some catalytic properties of phosphoglucomutase from maize leaves

Phosphoglucomutase (EC 2.7.5.1, PGM) was purified to homogeneity from maize (Zea mays L.) leaves. The enzyme had specific activity 11. 7 U/mg protein and molecular mass (determined by gel-chromatography) of 133 +/- 4 kD. The molecular mass of PGM subunits determined by SDS-electrophoresis was 66 +/- 3 kD. The enzyme had Km for glucose-1-phosphate and glucose-1,6-diphosphate of 20.0 +/- 0.9 and 16.0 +/- 0.8 &mgr;M, respectively. Concentrations of glucose-1-phosphate and glucose-1,6-diphosphate above 3 and 0.4 mM, respectively, cause substrate inhibition. The enzyme activity was maximal at pH 8.0 and temperature 35 degreesC. Magnesium ions activate the enzyme and manganese ions inhibit it. 3-Phosphoglycerate is an uncompetitive inhibitor of the enzyme (Ki = 1.22 +/- 0.05 mM). Fructose-6-phosphate, 6-phosphogluconate, and ADP activate PGM, whereas ATP, UTP, and AMP inhibit the enzyme. Citrate was also a potent inhibitor, inhibitory effects of isocitrate and cis-aconitate being less pronounced.     

Metabolism of glucose by unicellular blue-green algae

Summary A facultative photo- and chemoheterotroph, the unicellular bluegreen alga Aphanocapsa 6714, dissimilates glucose with formation of CO₂ as the only major product. A substantial fraction of the glucose consumed is assimilated and stored as polyglucose (probably glycogen). The oxidation of glucose proceeds through the pentose phosphate pathway. The first enzyme of this pathway, glucose-6-phosphate dehydrogenase, is partly inducible. In addition, the rate of glucose oxidation is controlled, at the level of glucose-6-phosphate dehydrogenase function, by the intracellular level of an intermediate of the Calvin cycle, ribulose-1,5-diphosphate, which is a specific allosteric inhibitor of this enzyme. As a consequence, the rate of glucose oxidation is greatly reduced by illumination, an effect reversed by the presence of DCMU, an inhibitor of photosystem II.Two obligate photoautotrophs, Synechococcus 6301 and Aphanocapsa 6308, produce CO₂ from glucose at extremely low rates, although their levels of pentose pathway enzymes and of hexokinase are similar to those in Aphanocapsa 6714. Failure to grow with glucose appears to reflect the absence of an effective glucose permease. A general hypothesis concerning the primary pathways of carbon metabolism in blue-green algae is presented.Abbreviations A (U)DPG ADP-glucose or UDP-glucose - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - G_(int.) intracellular glucose - F-6-P fructose-6-phosphate - 6-PG 6-phosphogluconate - Ru-5-P ribulose-5-phosphate - RUDP ribulose-1,5-diphosphate - PGA 3-phosphoglycerate - GAP glyceraldehyde-3-phosphate     

Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.     

Low glucose-1, 6-bisphosphate and high fructose-2, 6-bisphosphate concentrations in muscles of patients with glycogenosis types VII and V

The level of glucose-1, 6-bisphosphate, a potent allosteric activator of phosphofructokinase, was markedly decreased in muscles of patients with glycogenosis type VII (muscle phosphofructokinase deficiency) and type V (muscle phosphorylase deficiency). Glucose-1-phosphate kinase activity in muscle was virtually absent in a patient with glycogenosis type VII, whereas it was normal in a patient with type V glycogenosis. Glucose-1-phosphate level was increased in type VII glycogenosis, whereas it was decreased in type V glycogenosis. Another activator of phosphofructokinase, fructose-2, 6-bisphosphate was increased in muscles of patients with both types of glycogenosis although it was much higher in type VII than in type V. This finding may be partly related to the difference of fructose-6-phosphate concentrations. The results suggest that phosphofructokinase would contribute to the major glucose-1-phosphate kinase activity in normal human muscle and would also form a kind of self-activating system.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.