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J A Bassham

Publications and source records attributed to J A Bassham.

At least 55 records · Page 3Linked to original sources
PubMed1976

Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714.

Cells of Aphanocapsa 6714 were subjected to alternating ligh-dark periods (flashing-light experiments). The corresponding activation (in the light) and inactivation (in the dark) of the reductive pentose cycle was measured, in vivo, from initial rates of 14CO2 incorporation and also by changes in the total concentration of 14C and 32P in soluble metabolites. Two principle sites of metabolic regulation were detected: (i) CO2 fixation was inactivated 15 to 20 s after removal of the light source, but reactivated rapidly on reentering the light; (ii) hydrolysis of fructose-1,6-diphosphate (FDP) and sedoheptulose-1,7-diphosphate (SDP) by their respective phosphatase(s) (FDP + SDPase) was rapidly inhibited in the dark but only slowly reactivated in the light. The time required for reactivation of FDP + SDPase, in the light, was on the order of 20 to 30 s. As a consequence of the timing of these inactivation-reactivation reactions, newly fixed CO2 accumulated in the FDP and SDP pools during the flashing-light experiments. Changes in the concentrations of the adenylate pools (mainly in the levels of adenosine 5'-triphosphate and adenosine diphosphate) were fast in comparison to the inactivation-reactivation reactions in the reductive pentose cycle. Thus, these regulatory effects may not be under the control of the adenylates in this organism. The activation of CO2 fixation in the light is at least in part due to activation of phosphoribulokinase, which is required for formation of ribulose-1,5-diphoshate, the carboxylation substrate. Phosphoribulokinase activity in crude extracts was found to be dependent on the presence of strong reducing agents such as dithiothreitol, but not significantly dependent on adenylate levels, although adenosine 5'-triphosphate is a substrate.

Adenosine Diphosphate↗
PubMed1975

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.

Adenosine Triphosphate↗
PubMed1975

Regulation of ribulose 1,5-diphosphate carboxylase by substrates and other metabolites: further evidence for several types of binding sites.

Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mm Mg(2+) and low CO(2) concentrations by its substrates (RuDP and CO(2)) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO(3) (-) and Mg(2-), and this inactivity persists for 20 minutes or longer after 1 mm HCO(3) (-) and 10 mm Mg(2+) are added. Maximum activity requires that the preincubation mixture also include either 0.01 mm 6-PGluA or 0.5 mm NADPH.When the enzyme, following preincubation with HCO(3) (-) and Mg(2+), is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The Ki value for 6-PGluA is 0.02 mm and the Ki value for FDP is 190 mum. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition.In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A(1), A(2), and A(3). RuDP binds very tightly to site I (in the absence of Mg(2+) or HCO(3) (-)), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg(2+) and 1 mm HCO(3) (-). Mg(2+) and HCO(3) (-) (or CO(2)) bind to site A(3) (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A(1) and NADPH at site A(2) during preincubation with Mg(2+) and HCO(3) (-). With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A(1).The physiological role of this regulation is discussed, particularly with respect to protection of "C-3" plants against oxidation of RuDP to phosphoglycolate.

Journal Article↗
PubMed1975

Metabolism in normal and virus-transformed chick embryo fibroblasts as observed with glucose labeled with 14C and tritium and with tritium-labeled water.

Glucose metabolism in normal and virus-transformed chick embryo fibroblast cells in culture was observed by allowing the cells to metabolize [U-14C]glucose plus glucose labeled with tritium in the C-1, C-3, and C-6 positions. Similarities and differences between normal and transformed cells were observed and measured. Both normal and transformed cells are found to metabolize about 20% of the glucose via the oxidative pentose phosphate cycle, with the rates being about twice as much for transformed cells as for normal cells under the chosen conditions. Nevertheless, the ratio of glucose metabolized via oxidative pentose cycle to the net flow of that metabolized directly to fructose 6-phosphate is about the same in normal and transformed cells. Although the rate of flow of [14C]glucose into the tricarboxylic acid cycle intermediates and amino acids derived from them appears to be the same in normal and transformed cells, the rate of tritium incorporation from H3HO into these intermediates seems to be much higher in normal cells.

Animals↗
PubMed1974

Activation of ribulose 1,5-diphosphate carboxylase by nicotinamide adenine dinucleotide phosphate and other chloroplast metabolites.

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 10 mm MgCl(2) and 1 mm bicarbonate in the absence of ribulose 1,5-diphosphate, can be further activated about 170% with 0.5 mm NADPH present in the preincubation mixture. NADP(+), NADH, and NAD(+) are ineffective. The activation by NADPH is comparable to that previously seen with 0.05 to 0.10 mm 6-phosphogluconate in that these specific preincubation conditions are required, but the effects of NADPH and 6-phosphogluconate are not additive. Moreover, where higher concentrations of 6-phosphogluconate inhibited the enzyme, higher concentrations of NADPH give a greater activation, saturating at about 1 mm and 200%. Under the specified conditions of preincubation, fructose 1,6-diphosphate has an activation curve similar to that of 6-phosphogluconate, peaking at 0.1 mm and 70%. Above this level, activation decreases, and inhibition is seen at still higher concentrations. Other metabolites tested produced smaller or no effects on the enzyme activity assayed under these conditions. When either reduced NADP or 6-phosphogluconate are present in the preincubation mixture, it becomes possible to determine the Km for bicarbonate using a Lineweaver-Burk plot, and the Km for bicarbonate under these conditions is 2.8 mm, corresponding to 0.3% CO(2) at pH 7.8 and 25 C.

Journal Article↗
PubMed1973

Dynamics of metabolism of normal and virus-transformed chick cells in culture.

Application of steady-state tracer technique to normal and transformed cells in tissue culture allows quantitation of intracellular pool sizes of many metabolites and determination of rate of carbon flow along diverse paths. Using a unique apparatus to control the environmental conditions, we show that the glucose carbon flow into tricarboxylic acid cycle intermediates and amino acids is unchanged upon transformation. The increased glycogen formation and glycolysis varies with the glucose concentration in the medium, correlates with the faster glucose transport of transformed cells, and cannot be explained by a difference in growth rate alone.

Animals↗
PubMed1973

Activation and inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate.

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl(2) in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 mum 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed.

Journal Article↗
PubMed1973

Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa.

In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% (14)CO(2) or 0.05% (14)CO(2) in air, gassing with 100% O(2) results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% (14)CO(2) than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O(2).

Journal Article↗
PubMed1973

Efficiency of energy conversion by aerobic glucose metabolism in Aphancapsa 6714.

Efficiency of adenosine triphosphate (ATP) formation from glucose oxidation in Aphanocapsa 6714 was estimated by quantitative measurement of phosphorylated intermediary metabolites and glycogen (polyglucose) formed from (14)C-glucose. P/2e ratios based on oxygen uptake ranged from 2.62 to 3.08, whereas those based on (14)CO(2) evolution ranged from 1.66 to 1.72. The synthesis of glycogen, which is the dominant energy-consuming process in resting cells exposed to exogenous glucose, was almost totally inhibited under anaerobic conditions, and the cellular concentration of ATP decreased steadily. Thus, both net synthesis of ATP and the steady-state concentration of ATP are obligatorily linked to respiration in this heterotrophic unicellular blue-green alga.

Adenosine Diphosphate↗
PubMed1973

Kinetics of glucose incorporation by Aphanocapsa 6714.

Photoautotrophic metabolism of CO(2) was compared with glucose metabolism in the facultative unicellular blue-green alga, Aphanocapsa 6714. Glucose-fed cells incorporated more (14)C into phosphorylated sugar intermediates of the reductive and oxidative pentose phosphate cycles than autotrophic cells. The relative increases were: 140-fold in dark cells; 32-fold in dichlorophenylmethylurea (DCMU)-inhibited cells; and 16-fold in cells assumilating glucose during photosynthetic carbon reduction. On the other hand, incorporation of (14)C from glucose into 3-phosphoglycerate and the amino acid pools of glutamate and aspartate was reduced in dark cells. Rates of protein synthesis in dark and DCMU-inhibited cells were reduced 50 and 80% compared to photoautotrophic cells. In cells assimilating glucose during photosynthesis, rates of (14)C incorporation into the two amino acids and protein were the same as in photoautotrophic cells. Chase experiments, using an excess of (12)C-glucose and CO(2), revealed slow turnover of carbon in dark cells and intermediate turnover rates in DCMU-inhibited cells, when compared to cells assimilating glucose during photosynthesis.

Aspartic Acid↗
PubMed1972

Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts.

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO(2) in the chloroplasts, nor does it stimulate CO(2) fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO(2) fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.

Journal Article↗