Stringent control of glycolysis in Escherichia coli.

When $\text{Escherichia}\text{coli}\text{CP78}\text{(}\text{rel}{}^{\mathrm{+}}$) growing on glucose was starved for isoleucine by the addition of valine, the intracellular levels of fructose 6-phosphate, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were abruptly decreased to one-half, but those of glucose 6-phosphate and ATP remained constant. In contrast, this was not the case with CP79($\text{rel}{}^{\mathrm{?}}$). Chloramphenicol released the response observed in CP78. These results suggest that the glycolytic activity is also under the stringent control. Since only glucosephosphate isomerase[EC 5.3.1.9] was significantly inhibited by guanosine 5′-diphosphate 3′-diphosphate among several glycolytic enzymes tested, the enzyme might be responsible for the decrease observed in CP78.     

Energy metabolism in developing Ascaris lumbricoides eggs. II, The steady state content of intermediary metabolites.

The steady state levels of intermediary metabolites were measured in freeze clamped, developing, dormant, and activated infective Ascaris lumbricoides eggs. The $\text{[ATP]}\text{[ADP]}$ ratio is low in the developmental stages and rises sharply in the dormant egg; on activation of the dormant egg the $\text{[ATP]}\text{[ADP]}$ ratio falls. The levels of the phosphorylated glycolytic intermediates of acetyl-CoA and of isocitrate do not change markedly during development, but the levels of lactate, citrate, 2-oxoglutarate, glutamate, succinate, and malate all show significant changes in the developing, dormant, and activated egg. The dormant egg also appears to be characterized by a low cytoplasmic redox potential.     

Stereospecific activity of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine and comparison of analogs in the degranulation of platelets and neutrophils

1-O-Hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet activating factor) stimulated the degranulation of rabbit platelets and human neutrophils, whereas the enantiomer, 3-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-1-phosphocholine, was inactive. The analogs compared had the following relative potencies in degranulating platelets and neutrophils: 1-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine > 1-O-hexadecyl-2-O-ethyl-$\text{sn}$-glycero-3-phosphocholine >$\text{rac}$-1-O-octadecyl-2-O-ethylglycero-3-phosphocholine = 1-O-hexadecyl-2-O-methyl-$\text{sn}$-glycero-3-phosphocholine >$\text{rac}$-1-O-dodecyl-2-O-ethyl-glycero-3-phosphocholine. The deacetylated compound, 1-O-hexadecyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine, and 1-O-hexadecyl-2,2-dimethylpropanediol-3-phosphocholine were inactive. The active analogs selectively desensitized the response to each other in the neutrophils. It is suggested that these compounds may activate cells through interaction with a stereospecific receptor.     

Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides

A highly purified and active cytochrome $\text{b}$-$\text{c}$₁ complex has been isolated from the chromatophores of the photosynthetic bacteria $\text{Rhodopseudomonas}\text{sphaeroides}$ R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome $\text{c}$ with a turnover number of 1500 per minute at 23° based on cytochrome $\text{c}$₁. It contains 8.3 nmoles of cytochromes $\text{b}$ and $\text{c}$₁ per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis.     

Interdependence of glucose and arginine catabolism in Streptococcus faecalis R. ATCC 8043

The growth of $\text{Streptococcus faecalis}$ R.ATCC 8043 was found to be dependent on the simultaneous presence of both glucose and arginine, the molar ratio of utilization of these nutrients being 1:1. The growth coefficient (Y-glucose or Y-arginine) was near about 30 during exponential phase suggesting the generation of 3 moles of ATP during glycolysis. Glycolytic activity in cells was directly proportional to the intracellular pool of either arginine or citrulline and in aged cells the loet glycolytic activity could be restored by the addition of arginine to thecell suspension. Cell free extract of $\text{S}$. $\text{faecalis}$ was found to transfer phosphate group from carbamyl phosphate (a catabolic product of arginine) to glucose.     

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. II. Site and mode of glycolytic activation during Ca2+-loading

Human red cells (RBC) respond to moderate Ca²⁺-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca²⁺-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase ($\text{GAPDH}\text{PGK}\text{)-steps}$. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca²⁺-extrusion rate and not to the loaded Ca²⁺. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca²⁺-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the $\text{GAPDH}\text{PGK}$ equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. $\text{NAD}\text{NADH}$ ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.     

Potent hypotensive activity of 1-O-hexadecyl-2-O-acetyl-sn-glycero-3-phosphocholine in spontaneously hypertensive rat

Chemically synthesized 1-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine possessed the most potent hypotensive activity compared with bradykinin, prostagrandin E₂ and I₂ when 5 nano moles/kg body weight of each drug were administered intravenously in spontaneously hypertensive rat. The potency and the duration of hypotensive activity of 1-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine were dose dependent. Exogenous norepinephrine or angiotensin II showed pressor activity during the hypotensive action of 1-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine, but did not disturb the hypotensive pattern of this ether lipid. These may suggest that 1-O-alkyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine plays an important role for the regulation of blood pressure.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

125I labelled inulin: a convenient marker for deposition of liposomal contents in vivo

The utility of an iodinated derivative of inulin (¹²⁵I-tyraminyl-inulin, ¹²⁵ITI) for reporting $\text{in vivo}$ tissue distributions of liposomal contents is described. It is shown, employing a rat model, that this probe satisfies the criteria that the free form is rapidly cleared from the circulation and excreted, whereas ¹²⁵ITI encapsulated in large unilamellar vesicle (LUV) systems and subsequently taken up in various tissues exhibits a long (>3 days) retention time. Further, high specific activities (>1 μCi per μ1) are easily achievable, allowing low LUV dose levels (≤2.5 μmole phospholipid/kg body weight) to be employed. Minimal tissue workups for quantitation of ¹²⁵ITI distributions are required. It is concluded that from criteria of sensitivity, expense and simplicity, ¹²⁵ITI is a most convenient probe for characterizing liposome deposition $\text{in vivo}$.     

Studies on (Na+ + K+)-activated ATPase. XLIV. Single phosphate incorporation during dual phosphorylation by inorganic phosphate and adenosine triphosphate

$\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can be phosphorylated by its substrate ATP as well as by its product inorganic phosphate. The maximal capacity for phosphorylation by either of these two substances is one mol phosphate per mol enzyme. In order to investigate whether the enzyme molecule possesses only one phosphorylation site common to ATP and P_i, or two phosphorylation sites, one for ATP and one for P_i, dual phosphorylation of the enzyme has been carried out. Under conditions, which are maximally favourable for each type of phosphorylation, successive phosphorylation by P_i and ATP leads to a maximal incorporation of only one mol phosphate per mol enzyme. The phosphorylation capacity for ATP decreases by the same amount as the P_i-phosphorylation level increases, without an effect on the apparent affinity for ATP.The results can be explained by assuming either a single common phosphorylation site for P_i and ATP, or a conformational change of the enzyme following phosphorylation by P_i, which excludes phosphorylation by ATP.     

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcus mutans and its apparent identity with intracellular invertase.

An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of $\text{Streptococcus}\text{mutans}$ 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a K_m for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (K_i = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.     

Modulation of 25-hydroxyvitamin D3-24-hydroxylase by aminophylline: a cytochrome P-450 monooxygenase system.

The enzymatic activity of human erythrocyte pyruvate kinase was found to decrease on incubation of the purified enzyme with red blood cell ghosts, ATP and cAMP. If $\text{[}{}^{32}\text{P]γATP}$ was used radioactivity was found associated with the protein after gel electrophoresis. Various effectors protected the enzyme against phosphorylation. Treatment of the modified enzyme with a protein phosphatase restored enzymatic activity and also caused the loss of the radioactive label. Modification of the pyruvate kinase in this way altered the affinity of the enzyme for one of its substrates (phosphoenolpyruvate), but the binding of the other substrate (ADP) was unaffected.     

Flux regulation in glycogen-induced oscillatory glycolysis in cell-free extracts of Saccharomyces carlsbergensis

To localise the controlling point of the glycolytic system, the temporal changes of concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) step; a stationary state with all glycolytic intermediates having concentrations oscillating at nearly stationary mean values; and a depletion state at which the intermediates before the GAPDH step are being depleted due to the exhaustion of glycogen. In all these states, the mean ethanol production rate and the concentration of ATP and the intermediates beyond the GAPDH-step are maintained fairly constant, while the glycogen consumption rate and intermediate concentrations of the upper part of the glycolytic system change considerably: the glycogen consumption rate varies 4-fold and fructose-bis-phosphate concentration more than 10-fold. Doubling of the initial glycogen concentration and the addition of a great excess of fructose-bis-phosphate do not affect the ethanol production rate and the mean glycerate-3-phosphate (3-PGA) and pyruvate levels. By contrast, ethanol production was accelerated by an increase of the net ATP consumption rate resulting from either the addition of apyrase or by substitution of trehalose for glycogen. Neither the mean absolute ATP level nor the adenylate energy charge were measurably affected, however all this data can be interpreted in terms of a very strong stoichiometric regulation and stabilization of the lower part of the glycolytic system.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

A path analysis of the causal relationships between red cell glycolytic intermediates, ADP and ATP in sickle cell anemia

The statistical relationships among the glycolytic intermediates (GI)) of the Embden-Meyerhof pathway, adenine nucleotides (ANs) and various hematological measures were estimated for 34 sickle cell anemia patients. Heterogeneity in linear and quadratic regressions of hemoglobin and hematocrit, both singly and jointly, on the GI and AN variables implied 1) that any single formula to standardize optical density measures of the GIs and ANs on a per gram hemoglobin or per liter cell water basis would not uniformly remove hemoglobin and hematocrit effects: 2) that ignoring significant hematological effects could bias the estimates of correlation among GIs and ANs; and 3) that hemoglobin and hematocrit measures do not reflect the same source of variability. The correlations among the GIs and ANs, after adjustment for hematological variability, were analyzed by path analysis to determine which of five proposed path models for cause and effect relationships were compatible with the data. AMP had a greater influence on ADP (coefficient of determination (CD) = 23%) than all the GIs together, while G6P and ADP influenced ATP variability the most (CD = 33% and 12%). The contributions of unknown factors to ADP and ATP variability were large for all models (CD = 56--77%) possibly due to stress of sickle cell disease. The path model with AMP and the four GIs (G6P, F6P, FDP, DHAP) influencing ADP variation, and the same GIs and ADP influencing ATP was the model most compatible with the data.     

ESR studies on the orientation of cholesteryl ester in phosphatidylcholine multilayers

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester ($\text{I}$) and 1 : 14 ester ($\text{II}$). The nitroxide label of $\text{I}$ is aligned in the membrane with a very large angle of tilt (47° ± 1.5°) with respect to the normal to the membrane surface; $\text{II}$ does not show such a tilt. $\text{I}$ gives spectra corresponding to immobilized label while $\text{II}$ gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in $\text{I}$ and $\text{II}$ are not present at the phosphatidylcholine-water interphase.The data supports a ‘horseshoe’ configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.     

Photosynthetic electron transport,ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica

Isolated heterocysts of the N₂-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.     

Improved preparation and assay and some characteristics of Cl−-ATPase activity from Limonium vulgare

We present a revised method for the preparation, storage and assay of the Cl^?-ATPase activity responsible for salt secretion in Limonium vulgare. Altering the centrifugation step improved the yield and a linked-enzyme assay for ADP provided a sensitive method for continuous monitoring of Cl^?-ATPase activity. The activity of untreated membranes was low but fairly stable. Treatment with detergent gave strong stimulation of Cl^?-ATPase activity but caused a rapid decline in activity with time. $\text{V}{}_{\mathrm{<mtext>app</mtext>}}$ was approx. 6.5 μmoles/h per mg protein and $\text{K}{}_{\mathrm{<mtext>m,\; app</mtext>}}$ for ATP between 0.1 and 0.2 mM. Cl^? stimulated the activity up to a maximum at 0.13 M Cl^?, and the pH optimum was around 6.3 to 6.4.     

Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Microtubule-associated protein 2 (MAP 2) from the rat brain was phosphorylated by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. The apparent Km for MAP 2 of Kinase II was 0.2 μ$\text{M}$. The maximum incorporation of phosphate into MAP 2 by the action of Kinase II was about 5 mol of phosphate per mol of MAP 2, while that by the action of cAMP-dependent protein kinase was about 3 mol of phosphate per mol of MAP 2. When microtubule-associated proteins were incubated with both Kinase II and cAMP-dependent protein kinase together, about 7 mol of phosphate were incorporated into 1 mol of MAP 2.