Stimulation of Spermatid Phosphofructokinase by Fructose 2,6-Bisphosphate from Rat Testes

Effect of fructose 2, 6-bisphosphate on 6-phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) in spermatid extract from rat testes was studied. Fructose 2, 6-bisphosphate stimulated the enzyme greatly by increasing its affinity for fructose 6-phosphate and relieving the inhibition by ATP. Fructose 2, 6-bisphosphate (0.8 μM) was required for 50% activation of 6-phosphofructokinase (PFK). In addition, fructose 2, 6-bisphosphate, AMP and fructose 6-phosphate acted cooperatively to stimulate the activity of PFK. This stimulation may play an important role in the regulation of glycolysis in spermatids of rat testes.     

Relationship between Fructose 2,6-Bisphosphate and Carbohydrate Metabolism in Darkened Barley Primary Leaves

Initial dark fructose 2,6-bisphosphate levels in 10-day-old barley (Hordeum vulgare L.) leaves increased when the photosynthetic period was lengthened, when the temperature during the prior photosynthetic period was reduced, and following leaf excision. These treatments also increased the leaf sucrose concentration. Conversely, a decrease in dark fructose 2,6,-bisphosphate occurred during extended darkness, with increasing leaf age and when photosynthate in the leaf was reduced by earlier low light treatments. These variations in fructose 2,6-bisphosphate content correlate with known changes in dark respiration. These findings suggest, but do not conclusively prove, a causal relationship between dark fructose 2,6-bisphosphate levels and dark respiration rates.     

Quantity and Kinetic Properties of Ribulose 1,5-Bisphosphate Carboxylase in C3, C4, and C3-C4 Intermediate Species of Flaveria (Asteraceae)

The kinetic properties of ribulose 1,5-bisphosphate carboxylase(RuBPC) appear to have been modified during evolution of photosynthesisto adjust to changes in substrate availability. C₄ plants areconsidered to have a higher concentration of CO₂ available toRuBPC than C₃plants. In this study, the K_m(CO₂ and catalyticcapacity (k_cat) of RuBPC and the ratio of RuBPC protein to totalsoluble protein from several Flaveria species, including C₃,C₃-C₄ intermediate, and C₄ species, were determined. The C₃and intermediate species had similar K_m(CO₂) values while theC₄ species on average had higher K_m(CO₂) values. The mean ratioof K_cat/K_m for species of each group was similar, supportingthe hypothesis that changes in K_m and K_cat, are linked. Theallocation of total soluble protein to RuBPC was lowest in theC₄ Flaveria species, intermediate in the C₃-C₄ species, andhighest in the C₃ species. The results suggest that during evolutionof C₄ photosynthesis adjustments may occur in the quantity ofRuBPC prior to changes in its kinetic properties. (Received January 4, 1989; Accepted April 11, 1989)     

Interrelations between C4 Ketogenesis, C5 Ketogenesis, and Anaplerosis in the Perfused Rat Liver

We investigated the interrelations between C₄ ketogenesis (production of β-hydroxybutyrate + acetoacetate), C₅ ketogenesis (production of β-hydroxypentanoate + β-ketopentanoate), and anaplerosis in isolated rat livers perfused with ¹³C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C₄ and C₅ ketone bodies and of related acyl-CoA esters reveal that C₄ and C₅ ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C₅ ketogenesis from heptanoate is much lower than the rate of C₄ ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C₅ ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of β-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.The regulation of the metabolism of C₄ ketone bodies, i.e. β-hydroxybutyrate (BHB)² and acetoacetate (AcAc) has been extensively investigated in vivo in isolated livers, hepatocytes, and subcellular preparations (for reviews, see Refs. 1–4). In contrast, very little information is available on the metabolism of C₅ ketone bodies, i.e. β-hydroxypentanoate (BHP) and β-ketopentanoate (BKP), which are known in the clinical literature as 3-hydroxyvalerate and 3-ketovalerate (5, 6). The C₅ ketone bodies are formed in liver from the partial oxidation of odd-chain fatty acids (see Fig. 1, center column). C₅ ketogenesis uses the same enzymes of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) cycle as C₄ ketogenesis. The counterpart of HMG-CoA in C₅ ketogenesis is 3-hydroxy-3-ethylglutaryl-CoA (HEG-CoA). We only found one report on the formation of [¹⁴C]HEG-CoA in liver extract incubated with propionyl-CoA and [1-¹⁴C]acetyl-CoA (7).Open in a separate windowFIGURE 1.Scheme of C₄ ketogenesis and C₅ ketogenesis in the liver. Numbers refer to the following enzymes: 3-ketoacyl-CoA thiolase (1), HMG-CoA synthase (2), HMG-CoA lyase (3), and β-hydroxybutyrate dehydrogenase (4). The figure also shows the link between propionyl-CoA and the CAC via anaplerosis.Because odd-chain fatty acids are absent from the diet of non-ruminant mammals, body fluids contain only traces of C₅ ketone bodies. However, C₅ ketone bodies and hydroxyethylglutarate are found in body fluids of patients with disorders of the anaplerotic pathway, propionyl-CoA → methylmalonyl- CoA → succinyl-CoA, such as deficiency in propionyl-CoA carboxylase and methylmalonyl-CoA mutase as well as biotin or vitamin B12 deficiency (5, 6, 8). The formation of C₅ ketone bodies in these pathological states involves either the conversion of propionyl-CoA to BKP-CoA via 3-ketoacyl-CoA thiolase (Fig. 1, reaction 1) or the β-oxidation of odd-chain fatty acids synthesized in these patients (9) using propionyl-CoA as a primer (10).Like their C₄ counterparts, the C₅ ketone bodies are interconverted by mitochondrial BHB dehydrogenase (11). In peripheral tissues, C₅ ketone bodies are converted to propionyl-CoA (which is anaplerotic) + acetyl-CoA via 3-oxoacid-CoA transferase (12) and 3-ketoacyl-CoA thiolase. Peripheral tissues have a high capacity to utilize exogenous C₅ ketone bodies (13), especially heart, kidney, and brain, which have high activities of 3-oxoacid-CoA transferase (14, 15).Our interest in C₅ ketone body metabolism arose from an ongoing clinical trial where patients with long chain fatty acid oxidation disorders are treated with a diet containing triheptanoin (16, 17) instead of the classical treatment with the even-chain triglyceride trioctanoin. These patients suffer from muscle weakness and rhabdomyolysis, manifested by the release of creatine kinase in plasma. It was hypothesized that the accumulation of long chain acyl-CoAs and long chain acylcarnitines results in membrane damage with release of large and small molecules from cells. The leakage of small molecules would deplete intermediates of the citric acid cycle (CAC) which carry acetyl groups as they are oxidized. It was further hypothesized that boosting anaplerosis with a suitable substrate would compensate for the chronic cataplerosis and improve heart and muscle function. The catabolism of heptanoate yields propionyl-CoA, which can be used for anaplerosis in most tissues, and C₅ ketone bodies in liver. C₅ ketone bodies are converted to propionyl-CoA, which can be used for anaplerosis in peripheral tissues. The marked improvement of the patients'' conditions after switching from a trioctanoin- to a triheptanoin-based diet supported the hypothesis.After ingestion of meals containing triheptanoin as the only lipid component, both C₅ ketone bodies and C₄ ketone bodies accumulated in the plasma of patients that have been diagnosed with disorders of long chain fatty acid oxidation (16). This suggested that acetyl groups derived from heptanoate can be used for the synthesis of C₄ and C₅ ketone bodies. Alternatively, the accumulation of C₄ ketone bodies after triheptanoin ingestion might result from the inhibition of the utilization of C₄ ketone bodies in peripheral tissues by C₅ ketone bodies.The aim of the present study was to investigate the interaction between C₄ and C₅ ketogenesis in rat livers perfused with octanoate and/or heptanoate. To gain insight on the fates of the acetyl groups of both fatty acids and on the fate of the propionyl-CoA moiety of heptanoate, we conducted the experiments with a series of labeled substrates: [1-¹³C]octanoate, [8-¹³C]octanoate, [5,6,7-¹³C₃]heptanoate, [1-¹³C]heptanoate, and [¹³C₃]propionate. The outcome of the propionyl-CoA moiety of [5,6,7-¹³C₃]heptanoate and [¹³C₃]propionate was traced by measurements of anaplerosis and glucose labeling by mass isotopomer³ analysis (18). In previous studies on the metabolism of odd-chain fatty acids in liver or hepatocytes (19, 20), ketone bodies were assayed with BHB dehydrogenase. This assay does not differentiate C₄ from C₅ ketone bodies. In the present study we used gas chromatography-mass spectrometry to specifically assay C₄ and C₅ ketone bodies (13).     

Phosphoenolpyruvate Carboxylase Purified from Leaves of C3, C4, and C3-C4 intermediate species of Alternanthera: Properties at Limiting and Saturating Bicarbonate

Photosynthetica - Phosphoenolpyruvate carboxylase (PEPC) was purified from leaves of four species of Alternanthera differing in their photosynthetic carbon metabolism: Alternanthera sessilis (C3),...     

Phosphoenolpyruvate Carboxylase Purified from Leaves of C3, C4, and C3-C4 intermediate species of Alternanthera: Properties at Limiting and Saturating Bicarbonate

Phosphoenolpyruvate carboxylase (PEPC) was purified from leaves of four species of Alternanthera differing in their photosynthetic carbon metabolism: Alternanthera sessilis (C₃), A. pungens (C₄), A. ficoides and A. tenella (C₃-C₄ intermediates or C₃-C₄). The activity and properties of PEPC were examined at limiting (0.05 mM) or saturating (10 mM) bicarbonate concentrations. The V_max as well as K_m values (for Mg²⁺ or PEP) of PEPC from A. ficoides and A. tenella (C₃-C₄ intermediates) were in between those of C₃ (A. sessilis) and C₄ species (A. pungens). Similarly, the sensitivity of PEPC to malate (an inhibitor) or G-6-P (an activator) of A. ficoides and A. tenella (C₃-C₄) was also of intermediate status between those of C₃ and C₄ species of A. sessilis and A. pungens, respectively. In all the four species, the maximal activity (V_max), affinity for PEP (K_m), and the sensitivity to malate (K_I) or G-6-P (K_A) of PEPC were higher at 10 mM bicarbonate than at 0.05 mM bicarbonate. Again, the sensitivity to bicarbonate of PEPC from C₃-C₄ intermediates was in between those of C₃- and C₄-species. Thus the characteristics of PEPC of C₃-C₄ intermediate species of Alternanthera are intermediate between C₃- and C₄-type, in both their kinetic and regulatory properties. Bicarbonate could be an important modulator of PEPC, particularly in C₄ plants. This revised version was published online in August 2006 with corrections to the Cover Date.     

3-Hydroxybutyrate Aids the Recovery of the Energy State from Aglycaemic Hypoxia of Adult but Not Neonatal Rat Brain Slices

Abstract: The level of phosphocreatine (PCr) and the intracellular pH (pH_i) of superfused cortical brain slices from adult or 10-day-old rats were monitored using ³¹P NMR. When the glucose in the superfusing medium was replaced by 3-hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pH_i was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pH_i returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.     

Comparative Anatomy and Morphology of Leaves between C3 and C4 Species in Panicum

Anatomical and morphological structures of leaf blades werecompared between C₃ and C₄ species in Panicum. Inter-specificvariation of stomatal density, longitudinal vein density andmesophyll thickness was highly correlative either plus or minuswithin respective groups. The two groups could not be distinguishedby a single character, since the variation ranges overlappedeach other. However, the quantitative relations between veindensity and the other two characters differentiated the twogroups well. In C₃, stomatal density seemed to be a primaryfactor for regulating water balance, while in C₄ vein systemwas considered to be important for the regulation. The specieswith intermediate photosynthesis behaved similar to the C₃ species.In the C₃ group, correlative variation was observed betweenleaf width, leaf angle and the three characters mentioned above.Variation of light-receiving area due to the changes of widthand angle of leaf blades was considered to be one of the adaptivestrategies of this group. Increase of light-receiving area wasin connection with the thinning of leaves. On the other hand,in the C₄ correlations between length, width and angle of leaveswere low. Such loose character correlation may be achieved byits efficiency of CO₂ utilization and its well developed veinsystems. Besides, NAD-me type species tended to have relativelylower stomatal and vein densities as compared with the otherdecarboxylation types in this group. Panicum, photosynthesis, C₃, C₄, decarboxylation types, leaf, stomata, vein     

Quantitative Determination of RuBP Carboxylase-Oxygenase Protein in Leaves of Several C3 and C4 Plants

RuBP carboxylase-oxygenase protein in three C₃ species (Nicotianatabacum L., Solanum tuberosum L., Triticum aestivum L.) andthree C₄ species (Panicum miliaceum L., Panicum texanum Buckl.,Zea mays L.) was quantitatively determined by sucrose densitygradient centrifugation and by immunochemical assay using antibodyraised to crystallized tobacco leaf RuBP carboxylase-oxygenase.The C₃ species had 3- to 6-fold higher concentrations of RuBPcarboxylase-oxygenase than the C₄ species when expressed oneither a chlorophyll or a leaf area basis. The C₃ species alsoallocated a higher fraction of their total soluble protein tothis enzyme (from 25 to 60% for the C₃ species compared to 8to 23% for the C₄ species). There was no RuBP carboxylase-oxygenaseprotein or activity in the C₄ mesophyll cells, while the enzymeconstituted from 20 to 40% of the total soluble protein in theC₄ bundle sheath cells. A close correlation (r = +0·91)was found between catalytic activity and level of the enzymeprotein in the species examined.     

Effect of Wound Shock on Protein Synthesis in Leaves of C3 and C4 Plants

Wound stress causes an initial decrease in the uptake and subsequentincorporation of radiolabelled methionine in leaves of C₃ (peanutand soybean) and C₄ (sorghum and maize) plants. A four-hourincubation of excised leaves at 25C permits them to recoverfrom wounding and thereby facilitates monitoring of changesin protein synthesis caused by heat shock or other types ofenvironmental stress. (Received December 7, 1992; Accepted February 16, 1993)     

Carbon Isotopic Fractionation Does Not Occur during Dark Respiration in C3 and C4 Plants

The magnitude of possible carbon isotopic fractionation during dark respiration was investigated with isolated mesophyll cells from mature leaves of common bean (Phaseolus vulgaris L.), a C3 plant, and corn (Zea mays L.), a C4 plant. Mesophyll protoplasts were extracted from greenhouse-grown leaves and incubated in culture solutions containing different carbohydrate substrates (fructose, glucose, and sucrose) with known [delta]13C values. The CO2 produced by protoplasts after incubation in the dark was collected, purified, and analyzed for its carbon isotope ratio. From observations of the isotope ratios of the substrate and respired CO2, we calculated the carbon isotope discrimination associated with metabolism of each of these substrates. In eight of the 10 treatment combinations, the carbon isotope ratio discrimination was not significantly different from 0. In the remaining two treatment combinations, the carbon isotope ratio discrimination was 1[per mille (thousand) sign]. From these results, we conclude that there is no significant carbon isotopic discrimination during mitochondrial dark respiration when fructase, glucose, or sucrose are used as respiratory substrates.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Immunologically Distinct Forms of Adenylate Kinase in Leaves: Comparison of Subunit Size of Adenylate Kinase from C3 and C4 Plants

Kleczkowski, L. A. and Randall, D. D. 1987. Immunologicallydistinct forms of adenylate kinase in leaves: comparison ofsubunit size of adenylate kinase from C₃ and C₄ plants.—J.exp. Bot. 38: 1440–1445. Antibodies prepared against maize leaf adenylate kinase (E.C.2.7.4.3 [EC] ) cross-reacted with the enzyme isolated from leavesof both C₃ and C₄ plants. The immunoreaction was very specificas judged by the presence of a single band on Western immunoblotscontaining proteins from leaf extracts of several species. Themolecular weight (M₁) of adenylate kinase determined by meansof the immunoblotting was 29 kD and 27 kD for C₄ and C₃ species,respectively. For both C₃ and C₄ plants, the antibodies failedto precipitate all adenylate kinase activity in leaf extracts,while they were 100% effective in pelleting the enzyme frommaize mesophyll chloroplasts. This indicated the presence ofat least two immunologically distinct forms of adenylate kinasein leaves. It is suggested that the observed differences in molecular structure(M₁s) of adenylate kinase from C₃ and C₄ species might be responsiblefor distinct catalytic and functional properties of the enzymein these two groups of plants. The irrununologically-determinedoccurrence of distinct pools of adenylate kinase in leaves supportsprevious evidence obtained by means of subcellular fractionationstudies.     

Evolutionary transition from C3 to C4 photosynthesis and the route to C4 rice

Compared with C₃ plants, C₄ plants possess a mechanism to concentrate CO₂ around the ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts of bundle sheath cells so that the carboxylation reaction work at a much more efficient rate, thereby substantially eliminate the oxygenation reaction and the resulting photorespiration. It is observed that C₄ photosynthesis is more efficient than C₃ photosynthesis under conditions of low atmospheric CO₂, heat, drought and salinity, suggesting that these factors are the important drivers to promote C₄ evolution. Although C₄ evolution took over 66 times independently, it is hypothesized that it shared the following evolutionary trajectory: 1) gene duplication followed by neofunctionalization; 2) anatomical and ultrastructral changes of leaf architecture to improve the hydraulic systems; 3) establishment of two-celled photorespiratory pump; 4) addition of transport system; 5) co-option of the duplicated genes into C₄ pathway and adaptive changes of C₄ enzymes. Based on our current understanding on C₄ evolution, several strategies for engineering C₄ rice have been proposed to increase both photosynthetic efficiency and yield significantly in order to avoid international food crisis in the future, especially in the developing countries. Here we summarize the latest progresses on the studies of C₄ evolution and discuss the strategies to introduce two-celled C₄ pathway into rice.     

Arachidonic Acid, but Not Sodium Nitroprusside, Stimulates Presynaptic Protein Kinase C and Phosphorylation of GAP-43 in Rat Hippocampal Slices and Synaptosomes

Abstract: Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43-kDa growth-associated protein GAP-43, may contribute to the maintenance of hippocampal long-term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high-frequency stimulation of Schaffer collateral-CA1 synapses significantly increased the PKC-dependent phosphorylation of GAP-43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high-frequency stimulation on levels of immunoreactive phosphorylated GAP-43 was not observed when 4-amino-5-phosphonovalerate (50 µM), an N-methyl-d -aspartate (NMDA) receptor antagonist, was bath-applied during the high-frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates—arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)—were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC-specific GAP-43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 µM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP-43. The stimulatory effect of arachidonic acid on GAP-43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP-43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.     

A CO2-Flux Mechanism Operating via pH-Polarity in Hydrilla Verticillata Leaves With C3 and C4 Photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C₄-based, CO₂ concentrating mechanism (CCM) that concentrates CO₂ in the chloroplasts. Both C₃ and C₄ Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m⁻³), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO₂ flux mechanism (CFM), making HCO₃ ⁻ available for photosynthesis by conversion to CO₂. DIC at 10 mol m⁻³ completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m⁻³ DIC. At low irradiance (50 μmol photons m⁻² s⁻¹), where CCM activity in C₄ leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C₃ leaves decreased. In C₄ leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO₂ fixation in Hydrilla, also suppressed acidification and alkalization, especially in C₄ leaves. IA does not inhibit the C₄ CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H⁺-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP⁺ ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of Liver Glycogen Synthesis from [14C] Glucose and [14C] Lactate by Portal Arterial Glucose Difference in the Perfused Rat Liver

To study effects of the portal-arterial glucose difference on the hepatic glycogenesis, the liver was isolated from fasted rats and was bivascularly perfused. Thirty-five milliliters of Krebs-Ringer buffer (pH 7.4) with 2 mM glucose, 3 mM lactate, 20 ng/ml insulin, and [1-¹⁴C]glucose or [U-¹⁴C]lactate was recirculated at flow rates of 14 ml/min via the portal vein and 7 ml/min via the hepatic artery. Glucose was continuously infused at a rate of 27.75 μmol/min into the portal (P experiment) and the arterial cannula (A experiment), and the portal-arterial glucose gradients were + 1.98 and −3.96 mM. Perfusate glucose concentration was not different between the P and A experiments within 20 min. Perfusate lactate level was higher in the P experiment than in the A experiment at 20 min. Incorporation of radioactivity from [¹⁴C]glucosc into glycogen was higher in the P experiment than in the A experiment (0.245 ± 0.014%/20 min vs 0.175 ± 0.022%/20 min, P < 0.01), and not influenced by the addition of insulin. Incorporation of ¹⁴C from [¹⁴C]lactate into glycogen was not different between the P and A experiments, and was significantly increased with the addition of insulin. This activity, in the presence of insulin, was higher in the P experiment than in the A experiment (0.490 ± 0,028%/20 min vs 0.406 ± 0.025%/20 min, P < 0.05). These results suggest that the portal-arterial glucose difference has an important role in the regulation of hepatic glycogenesis from exogenous glucose and gluconeogenesis.