GLYCOLYSIS IN RAT PERITONEAL MAST CELLS

Glycolytic activity of rat peritoneal mast cells has been measured by the Cartesian ampulla diver technique. The rates of anaerobic glycolysis, expressed as CO₂ expelled from a bicarbonate medium, are 1.70 x 10^-6 µl and 1.43 x 10^-6 µl per cell per hour with and without glucose, respectively. The aerobic glycolysis rate in the presence of glucose, assuming the respiratory quotient to be 1, is 0.93 x 10^-6 µl CO₂ per cell per hour. It is pointed out that the anaerobic and non-respiratory aerobic carbon dioxide production by mast cells is much higher than the respiratory oxygen uptake reported previously. These values have been interpreted in terms of glucose utilization.     

THE ACTIVITIES OF BUTYRYLCHOLINESTERASE AND CARBONIC ANHYDRASE, THE RATE OF ANAEROBIC GLYCOLYSTS, AND THE QUESTION OF A CONSTANT DENSITY OF GLIAL CELLS IN CEREBRAL CORTICES OF VARIOUS MAMMALIAN SPECIES FROM MOUSE TO WHALE

Abstract— The question of a constant density of glial cells in mammalian cerebral cortex regardless of species was examined by surveying the cortical activities of two enzymes primarily localized to dial cells. The cortical activity of butyrylcholinesterase (EC 3.1.1.8) was essentially constant at a rate of approx. 0.1 μmol of butyrylthiocholine hydrolysed min^-1 g^-1 over the range of species from rat (brain wt., 1.6 g) to fin whale and sperm whale (brain wt., 6800 and 7800 g, respectively). Over the same range the activity of cortical acetylcholinesterase, a neuronal enzyme, decreases by a factor of 7. Thus, butyrylcholinesterase ranged from < 2 per cent (in small rodent brains) to approximately 10 per cent (in whale brain) of the cortical acetylcholinesterase activity. The cortical activity of carbonic anhydrase (EC 4.2.1.1) was constant at a rate of 6.2 (± 0.25) μmol of CO₂ evolved min^-1 g^-1 over the range of species from guinea-pig (brain wt., 4.75 g) to fin whale (brain wt., 6800 g). These data obtained by assaying the dehydration reaction were confirmed by limited assays of the esterase activity of the enzyme (with p-nitrophenylacetate as substrate) and agreed with limited, previously reported data for the hydration reaction. Thus, the circumstantial evidence strongly favoured a relative constancy of cortical glial cell density regardless of species. The rates of anaerobic glycolysis in the cerebral cortex of various species were also investigated. For six species from mouse (brain wt., 0.4 g) to beef (brain wt., 380 g) cortical anaerobic glycolysis varied only slightly in the range of 50–62 μmol of CO₂ evolved h^-1 g^-l, whereas cortical oxygen consumption for the same range of species decreased by a factor of 3. Previously frozen samples of beef cortex glycolysed at 35 per Cent of the rate of fresh (unfrozen) samples. Since identical rates were obtained for previously frozen samples of fin whale cerebral cortex, we concluded that the relative constancy of cortical anaerobic glycolysis could be extended to the range from mouse to whale and that this aspect of cortical metabolism is probably primarily glial in localization. Some implications of the latter conclusion for the proposed role of astrocytes as modulators of neuronal activity have been discussed.     

Effects of Various Concentrations of Carbon Dioxide on Respiration and Potassium Uptake in Barley Roots

Barley roots contain a CO₂ sensitive respiratory fraction which is inhibited in 50 per cent CO₂ and is partially restored upon subsequent exposure to air. The residual O₂ consumption occurring at CO₂ concentrations between 50 per cent and 95 per cent amounts to about 40 per cent of the O₂ uptake in air and can support K⁺ uptake for a limited time at a rate equal to or higher than occurs in air. Above 95 per cent CO₂ both O₂ and K⁺ uptakes decrease rapidly. 2,4-dinitrophenol (DNP), in the range of 10^?6 to 10^?5M, stimulates O₂ uptake by the roots in air. The stimulation is absent when roots are treated with DNP in 80 per cent CO₂, presumably because of the reduced demand for inorganic phosphate and phosphate acceptor at the lower respiratory level in high CO₂. In either air or CO₂, K⁺ uptake is strongly inhibited by DNP. A comparison of the respiratory and K⁺ uptake data indicates that O₂ consumption is a necessary requirement for the uptake process in high CO₂. Protoplasmic streaming in the root cells is rapidly stopped by high CO₂ although K⁺ uptake and O₂ consumption continue. The cation uptake mechanism in high CO₂ appears to be limited to the stationary cytoplasm. It is also possible that a similar mechanism may be involved in cation uptake in air.     

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis¹. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate², the same is not true for extracellular acid production and its relationship to glycolytic rate ^3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO₂, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate^- + H⁺ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO₂, hydration to H₂CO₃ and dissociation to HCO₃^- + H⁺ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification ⁶. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.     

THE EXCRETION OF CARBON DIOXIDE BY FROG NERVE

1. Quiescent sciatic nerve of the frog discharges CO₂ at the average rate of 0.00876 mg. CO₂ per gram of nerve per minute. 2. Sciatic nerve steeped one minute in boiling water discharges CO₂ at first at a low rate and after an hour and a half not at all. 3. Degenerated sciatic nerve discharges CO₂ at a slightly higher rate than normal living nerve does. 4. Connective tissue from the frog discharges CO₂ at an average rate of 0.0097 mg. per gram of tissue per minute. 5. Assuming that a nerve is composed of from one-half to one-quarter connective tissue the CO₂ output from its strictly nervous components is estimated to be at a rate of 0.008 mg. CO₂ per gram of nerve per minute. 6. Stimulated sciatic nerve increases the rate of its CO₂ output over quiescent nerve by about 14 per cent. When this number is corrected for strictly nervous tissue the rate is about 16 per cent. 7. The increased rate of CO₂ production noted on stimulation in normal sciatic nerves was not observed when they were boiled, blocked, or degenerated. It was also not observed with stimulated strands of connective tissue.     

CARBON DIOXIDE AS A FACILITATING AGENT IN THE INITIATION AND GROWTH OF BUBBLES IN ANIMALS DECOMPRESSED TO SIMULATED ALTITUDES

1. Rats killed in a variety of ways (broken neck, nembutal, anoxia, electrocution) may undergo extensive bubble formation when subsequently decompressed from atmospheric pressure to simulated altitudes of 50,000 feet. On autopsy at sea level, large numbers of bubbles are found throughout the vascular system in the majority of animals. These bubbles appear to originate in small vessels deep within muscular regions, later spreading widely in arterial and venous systems. Dead rabbits and frogs also bubble profusely on decompression. 2. Bubble formation in dead animals is attributed primarily to the accumulation of CO₂, derived from residual cellular respiration after death, and from anaerobic glycolysis with attendant decomposition of bicarbonates in blood and tissue fluids. If anaerobic glycolysis is inhibited by using sodium iodoacetate as a lethal agent, bubble formation is greatly reduced or lacking on subsequent decompression. 3. Experiments in vitro suggest that high concentrations of CO₂ favor bubble formation by reducing the degree of mechanical disturbance necessary. 4. Administration of CO₂ in high concentrations to living frogs lowers the minimum altitude (pressure equivalent) at which bubble formation occurs, with exercise, in untreated animals. Pre-treatment with CO₂ also reduces the degree of muscular activity necessary for bubbles to form in frogs at higher altitudes. 5. Analyses have been made of the gas content of bubbles taken directly from the large veins of decompressed frogs and rats. In living animals the figures obtained indicate rapid equilibration with gas tensions in the blood. Bubbles taken from decompressed dead rats may contain 60–80 per cent CO₂. 6. The bearing of these experiments on the mechanisms of bubble initiation and growth in normal living animals is discussed. Reasons are given for suggesting that CO₂, due largely to its high dissolved concentration in localized active regions, may be an outstanding factor in the initiation and early growth of bubbles which in later stages are expanded and maintained principally by nitrogen.     

THE EFFECT OF CARBON DIOXIDE TENSION ON TISSUE METABOLISM (RETINA)

The metabolism of rat retina was found to be sensitive to the concentration of the carbon dioxide-bicarbonate buffer system. Increasing the carbon dioxide from 1 per cent to 5 per cent at constant pH nearly doubled both respiration and glycolysis. Increasing the carbon dioxide at constant pH from 5 per cent to 20 per cent had no effect on glycolysis, but depressed the Q _OO2 from 31 to 19. In a medium containing glucose and the 1 per cent carbon dioxide-bicarbonate buffer, the addition of succinate increased the Q _OO2 from 12 to 26, without affecting glycolysis. In a medium containing glucose and phosphate, succinate had no significant effect.     

EXPERIMENTALLY INDUCED CHANGES IN THE BASE COMPOSITION OF THE RIBONUCLEIC ACIDS OF ISOLATED NERVE CELLS AND THEIR OLIGODENDROGLIAL CELLS

The effect of tricyano-amino-propene, a dimer of malononitrile, on the base composition of the RNA in isolated Deiters' nerve cells and their oligodendroglial cells has been studied using a microelectrophoretic method. Tri-a-p in a dose of 20 mg/kg has the effect of increasing the RNA and protein content per nerve cell by 25 per cent and decreasing the glia RNA by 45 per cent. The RNA base composition of the nerve cells from the control animals differs from that of their glial cells. The guanine of the nerve cell is significantly higher than that of the glia, but the content of cytosine is higher in the glia than in the RNA of nerve cell. The cytosine of nerve cells decreased significantly after tri-a-p administration. In the glial cells the cytosine showed a 20 per cent increase, and the guanine a 25 per cent decrease. Tri-a-p sharpened the difference in RNA composition already existing between the control nerve cells and their glial cells by almost 300 per cent for the guanine and by 400 per cent for the cytosine. The chemical and functional relationship between the nerve cell and its oligodendroglial cells is discussed.     

Aerobic and anaerobic respiration in the intact spinach chloroplast

Aerobic and anaerobic chloroplastic respiration was monitored by measuring ¹⁴CO₂ evolution from [¹⁴C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6-bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O₂ or in N₂ with nitrite or oxaloacetate as electron acceptors. The pathway of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N₂ were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O₂ was the best (2.4 nanomoles CO₂ per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N₂ with nitrite and oxaloacetate.     

Studies on the energy metabolism during anaerobic fermentation of glucose by baker's yeast

Summary As a result of the intimate association of ADP phosphorylation with alcoholic fermentation, resulting in the synthesis of 2 mole ATP per mole glucose fermented, it may be calculated that a minimum of 672 µcal heat development may be expected for every mm³ CO₂ developed during alcoholic fermentation. When all ATP produced would be fully de-phosphorylated to ADP + Pi (e.g. by ATP-ase activity) a maximum heat development of 1200 µcal per mm³ CO₂ could be expected.Using the LKB-Flow-Microcalorimeter for measurement of heat development and at the same time the Warburg technique for measuring CO₂ development during anaerobic glucose fermentation of a baker's yeast suspension, the heat development per mm³ CO₂ produced was calculated over a fermentation period of 90 min.Maintenance of strict anaerobic conditions in the Flow-Microcalorimeter vessel was complicated by diffusion of traces of oxygenvia the Teflon transport lines, resulting in excessive heat development values, not representative for the alcoholic fermentation. This problem could be circumvented by removal of traces of oxygen by means of addition of the enzyme glucose-oxidase.Poisoning the respiratory enzyme system of the yeast by addition of KCN or azide, or using respiratory-deficient mutants of the yeast also resulted in heat development values, inherent with alcoholic fermentation.The values obtained were very close to the minimum of 672 µcal per mm³ CO₂, at least during the initial phases of fermentation, indicating that ADP regeneration from ATP, essential for maintaining the high fermentation rate, is not primarily the result of ATP-ase activity, but must be due to participation of ATP in energy-requiring synthetic reactions.     

Der Einfluß von CO2 und pH auf die 32P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht

Summary The effect of CO₂ on the ³²P-labelling of polyphosphates and acid-soluble organic phosphates is studied in synchronously grown cultures of the green alga Ankistrodesmus braunii, using trichloroacetic acid treatment and acid hydrolysis for the fractionation of the phosphorus compounds.Three per cent CO₂ in nitrogen causes an inhibition of the labelling of polyphosphates but a marked increase of ³²P in organic phosphates, whereas oxygen (CO₂-free air) produces the reverse effect. Polyphosphates and ATP are the fractions most stimulated by O₂, while stable organic phosphates show the strongest inhibition. Labelling of nucleic acids is relatively indifferent to both oxygen and CO₂. Three per cent CO₂ in air causes the same distribution of ³²P-labelling as 3 per cent CO₂ in N₂. ³²P-labelling is strongly dependent on the pH of the medium. In the absence of CO₂, polyphosphate labelling is highest in the acidic range, whereas organic phosphates and ATP show optimum labelling and the highest percentage of the total ³²P in the alkaline pH range. The effect of CO₂ is strongest between pH 5 and 6, that of oxygen between pH 8 and 9. Apparently the pH of the medium exerts a considerable influence upon the phosphate metabolism inside the cells.Increasing concentration of CO₂ lead to the same change of ³²P-labelling in nitrogen as in air and to saturation at about 1 per cent CO₂ under the conditions used. The curves are in good agreement with those of O₂-evolution at increasing concentrations of CO₂, but they show completely different rates.Young cells respond to CO₂ and O₂ differently from cells in the photosynthetically most active stage. In young cells both gasses are less effective.The effect of CO₂ is explained by a strong increase in noncyclic photophosphorylation which can proceed only slowly in N₂. ATP-consumption connected with high rates of CO₂-fixation may be the reason for the low rates of ³²P-labelling in the polyphosphate fraction when CO₂ is present. The influence of external pH on ³²P-labelling is partly due to the pH-dependence of phosphate uptake, but the different response of several fractions to the pH of the medium suggests that the pH of the cytoplasm and possibly even the pH of the interior of the chloroplasts is affected by the external pH. The effect of O₂ in the absence of CO₂ or at low CO₂-concentrations is explained by the well-known inhibition of photosynthesis by oxygen. Increasing concentrations of CO₂ reverse this inhibition and correspondingly change the distribution of ³²P between the phosphate fractions. The change in sensitivity to CO₂ and O₂ with the cell age is consistent with the change in the rates of maximum photosynthetic CO₂-fixation.

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Herrn Prof. Dr. W. Schumacher zum 70. Geburtstag gewidmet.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

The Influence of Photosynthetic Factors and Metaholic Inhibitors on the Uptake of Phosphate in P-Deficient Scenedesmus

The cells used in the present investigation had a phosphate content of about 20 per cent as compared with the status in normal cultures. The uptake of phosphate during a period of 4 hours was determined at a pH of 6,5, kept constant with the aid of a citrate buffer. In the absence of CO₂, light increased the uptake of phosphate with saturation around 14,000 erg/cm²s. With 5 per cent CO₂ in the air the relationship was more complicated, and the uptake of phosphate must he related to more than one process during active photosynthesis. The inhibiting effect of CO₂ in air was noticeable already at low concentrations both in light and in darkness. With the system used, this supports earlier indications for internal recycling of orthophosphate, CO₂ was inhibiting also in nitrogen in the light. Selenate in a concentration of 2 mM gave a slight and rather irregular inhibition.—Anaerobiosis had no effect in the light but gave a large decrease in the dark.—DNP (0.1 mM) was somewhat more active in the dark than in the light. The lower concentrations tested had no effect in either case.—Menadione (0.1 mM) inhibited strongly, and more in illuminated than in non-illuminated cells.     

Respiratory metabolism in buckwheat seedlings

Young seedlings of buckwheat (Fagopyrum esculentum) respire in air with an RQ of unity. Analysis of respiratory substrates coupled with a study of the utilization of acetate-¹⁴C and glucose-¹⁴C suggest that both the Embden-Meyerhof-Parnas, tricarboxylic acid and pentose phosphate sequences participate in the total respiratory catabolism.

In anoxia CO₂ dropped to one third of the aerobic rate and ethanol accumulated to only about one half the rate of CO₂ output on a molar basis. Smaller amounts of lactate, succinate and free amino acids (particularly alanine and γ-aminobutyric acid) accumulated, carboxylic acids decreased and there were initial increased in pyruvate and α-ketoglutarate. The observed changes are consistent with residual tricarboxylic acid and pentose phosphate cycle activity in anoxia and may account for the excess CO₂ production over ethanol accumulation. CO₂, ethanol and lactate production did not account for all of the carbohydrate consumed in anoxia.

Relative rates of carbon loss were measured in air and in atmospheres containing 3.5%, 2.1%, 1.3% and 0.6% oxygen. The extinction point of anaerobic metabolism was 1.5%.

On return to air from anoxia the CO₂ output increased and the RQ rose from 0.8 to 1.0 over the first 2-hour period. Ethanol, lactate and succinate were consumed and other constituents returned to their previous aerobic level. Some of these changes suggest a rather slow resumption of tricarboxylic acid cycle activity on return to air.

Carbon loss as CO₂ in air was greater than the carbon loss as CO₂ at the extinction point. Carbon loss in anoxia as CO₂, ethanol and lactate was similar to carbon loss at the extinction point. Assessed in this orthodox manner buckwheat seedlings show no Pasteur effect but the complex nature of the changes in levels of metabolic substrates and intermediates do not allow firm conclusions to be drawn on the effects of oxygen on the rates of glycolysis and other respiratory processes.

     

THE PRODUCTION OF CARBON DIOXIDE BY NERVE

1. A modified Osterhout respiratory apparatus for the detection of CO₂ from nerve is described. 2. The lateral-line nerve from the dogfish discharges CO₂ at first with a gush for half an hour or so and then steadily at a lower rate for several hours. 3. Simple handling of the nerve does not increase the output of CO₂; cutting it revives gush. 4. The CO₂ produced by nerve is not escaping simply from a reservoir but is a true nervous metabolite. 5. The rate of discharge of CO₂ from a quiescent nerve varied from 0.0071 to 0.0128 mg. per gram of nerve per minute and averaged 0.0095 mg. 6. Stimulated nerve showed an increased rate of CO₂ production of 15.8 percent over that of quiescent nerve. 7. The results of these studies indicate that chemical change is a factor in nerve transmission.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Energy metabolism in hypoxic astrocytes: Protective mechanism of fructose-1,6-bisphosphate

The protective effects of fructose-1,6-biphosphate (FBP) during hypoxia/ischemia are thought to result from uptake and utilization of FBP as a substrate for glycolysis or from stimulation of glucose metabolism. To test these hypotheses, we measumed CO₂ and lactate production from [6-¹⁴C]glucose, [1-¹⁴C]glucose, and [U-¹⁴C]FBP in normoxic and hypoxic cultured astrocytes with and without FBP present. FBP had little effect on CO₂ production by glycolysis, but increased CO₂ production by the pentose phosphate pathway. Labeled FBP produced very small amounts of CO₂. Lactate production from [1-, and 6-¹⁴C]glucose increased similarly during hypoxic hypoxia; the increase was independent of added FBP. Labeled lactate from [U-¹⁴C]FBP was minimal. We conclude that exogenous FBP is not used by astrocytes as a substrate for glycolysis and that FBP alters glucose metabolism.     

The Influence of CO2 on the Metabolism of Rhizome Tissue in Iris pseudacorus

In vitro experiments showed that concentrations of CO₂ above 6 per cent inhibited succinate oxidation; 30–10 per cent of succinate oxidation may be blocked in the presence of 12 per cent CO₂. Storage of rhizome cores in CO₂ increased the levels of all the main acids except malic. The amino acids, aspartate, glutamate and alanine, also increased in amounts under these conditions. Cores fix CO₂ probably via the reaction phosphoenolpyruvate to oxaloacetate. Raised CO₂ levels increased the rate of diminution of carbohydrate, but carbon itself was conserved due to a reduction in CO₂ output.     

Effects of Light and Elevated Atmospheric CO(2) on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves

Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO₂ per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO₂ levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.

Diurnal measurements of RuBPCase (expressed in micromoles CO₂ per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO₃⁻ and Mg²⁺, but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO₃⁻ and Mg²⁺-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO₃⁻ and Mg²⁺-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.

These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO₂ and Mg²⁺.

     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.