Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Cholesterol esters and hydrolytic cholesterol esterase during Wallerian degeneration

To study the involvement of cholesterol esters in myelination and demyelination, we determined the concentration of free cholesterol and cholesterol esters and the activity of hydrolytic cholesterol esterase (sterol ester hydrolase; EC 3.1.1.13) in hen sciatic nerve during Wallerian degeneration. A progressive increase in the ratio of cholesterol ester to free cholesterol was observed in the degenerating nerve at 8, 16 and 32 days after nerve section. Hydrolytic cholesterol esterase activity decreased progressively in the degenerating nerves at the same time. In addition we measured the ratio of RNA to DNA, and the activity of the NADP⁺-dependent isocitrate dehydrogenase [L₈-isocitrate: NADP oxidoreductase (decarboxylating); EC 1.1.1.42] at 8, 16 and 32 days after nerve section. The RNA to DNA ratios decreased progressively in the degenerating nerves. NADP⁺-dependent isocitrate dehydrogenase increased in activity after nerve section, reaching a peak at 16 days.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Acute effects of ammonia on the enzymes of citric acid cycle in rat brain

Activities of the enzymes of citric acid cycle were determined along with aspartate and alanine aminotransferases and NADP⁺-isocitrate dehydrogenase in the brains of rats treated with an acute dose of ammonium acetate and compared with those of normal animals. Elevation in the activities of pyruvate, α-ketoglutarate and succinate dehydrogenases and citrate synthase was observed in hyperammonemic animals. The activities of malate, NADP⁺-isocitrate dehydrogenases and aminotransferases decreased under these conditions. The results suggest that ammonia toxicity might not be due to the depletion of α-ketoglutarate from citric acid cycle.     

Schistosomatium douthitti: carbohydrate metabolism in the adults.

Pathways of carbohydrate metabolism in the adults of Schistosomatium douthitti: were investigated. Histochemical reactions for adenosinetriphosphatase (EC 3.6.1.3) glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphogluconate dehydrogenase (EC 1.1.1.43), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), lactate dehydrogenase (EC 1.1.1.27, 1.1.2.3) isocitrate dehydrogenase (EC 1.1.1.41), succinate dehydrogenase (EC 1.3.99.1), malate dehydrogenase (EC 1.1.1.37), cytochrome oxidase (EC 1.9.3.1), and adenosine triphosphatase (EC 3.6.1.3) were found in the adult worms. Glycogen deposits occurred in the parenchyma.Low oxygen tension immobilized the worms. Tartar emetic, sodium cyanide reduced adult motility in vitro. Manometric experiments demonstrated a respiratory quotient of approximately one. Oxygen uptake was completely inhibited by tartar emetic and partially inhibited by sodium fluoracetate and sodium cyanide. Inhibition by sodium fluoroacetate was partially counteracted by citric acid in the medium.Adults demonstrated an oxygen debt following anaerobic incubation. A maximum of 52% of the glucose consumed under aerobic conditions was excreted as lactic acid. Under anaerobic conditions the amount of lactic acid excreted increased. Acids other than lactic acid were also released. Results indicate that although glycolysis is the major pathway, two additional aerobic pathways also exist, one which is cyanide sensitive and the other cyanide insensitive.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

Purification and characterization of a monomeric isocitrate dehydrogenase from the sulfate-reducing bacterium Desulfobacter vibrioformis and demonstration of the presence of a monomeric enzyme in other bacteria

NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) was purified to homogeneity from the sulfate-reducing bacterium Desulfobacter vibrioformis, and shown to be a monomeric protein with a molecular mass of 80 kDa. The pH and temperature optima were 8.5 and 45°C, respectively. The N-terminal amino acid sequence (Thr, Glu, Thr, Ile, Arg, Trp, Thr, X, Thr, Asp, Glu, Ala, Pro, Leu, Leu, Ala, Thr) showed similarity with that of other known monomeric isocitrate dehydrogenases. Catalytically active isocitrate dehydrogenase from D. vibrioformis was obtained by activity staining after SDS-PAGE and removal of SDS from the gel. This technique revealed a NADP⁺-dependent monomeric enzyme in other Desulfobacter spp., Desulfuromonas acetoxidans and Chlorobium tepidium. These findings imply that monomeric isocitrate dehydrogenases are present in distantly related bacteria and indicate an early evolution of monomeric isocitrate dehydrogenases in the bacterial lineage.     

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Purification and properties of NADP+:Isocitrate dehydrogenase from lactating bovine mammary gland

NADP⁺:isocitrate dehydrogenase has been purified to homogeneity from lactating bovine mammary gland. Purification was achieved through the use of affinity and DEAE-cellulose chromatography. The isolated enzyme gives one band when stained for protein or enzyme activity on discontinuous alkaline gel electrophoresis. The enzyme has a molecular weight of 55,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and a Stokes radius of 4.1 nm as measured by gel chromatography. The enzyme will not use NAD⁺ in place of NADP⁺ and has an absolute requirement for divalent cations. The apparent K_m values for dl-isocitrate, Mn²⁺, and NADP⁺ were found to be 8, 6, and 11 μm, respectively. The Mn²⁺-d_s-isocitrate complex is the most likely substrate for the mammary enzyme with a K_m of 3 μm. The properties of mammary NADP⁺:isocitrate dehydrogenase are compared with those of the homologous enzymes from pig heart and bovine liver, and its characteristics are discussed with respect to the function of the enzyme in lactating mammary gland.     

Partial Purification and Characterization of NADP-Isocitrate Dehydrogenase from Immature Pod Walls of Chickpea (Cicer arietinum L.)

NADP⁺-isocitrate dehydrogenase (threo-DS-isocitrate: NADP⁺ oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg²⁺ or Mn²⁺, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP⁺, dl-isocitrate, Mn²⁺, and Mg²⁺. Their K_m values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP⁺. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP⁺ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP⁺ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP⁺-IDH in situ is likely to be controlled by intracellular NADPH to NADP⁺ ratio as well as by the concentration of various substrates and products.     

Enzyme activities in the sheep placenta during the last three months of pregnancy

In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and ketone body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy.The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-Co A synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP⁺) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do no occur in the sheep placenta.It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.     

Caenorhabditis elegans and Ascaris suum: fragmentation of isocitrate lyase in crude extracts

It is well known that proteolysis often occurs after rupture of metazoan cells. Thus proteins isolated from extracts may not be representative of their native cellular counterparts. In the present research, extensive proteolysis was observed in crude extracts of the freeliving soil nematode Caenorhabditis elegans and the parasitic nematode Ascaris suum. Phenylmethylsulfonyl fluoride (PMSF) reduced the loss in activity of isocitrate lyase (EC 4.1.3.1), fumarase (EC 4.2.1.2), and citrate synthase (EC 4.1.3.7) in extracts of C. elegans but had little or no effect upon loss of malate synthase (EC 4.1.3.2). Catalase (EC 1.11.1.6) was stable. The loss of isocitrate lyase and citrate synthase was less pronounced in extracts of 22-day-old embryos of A. suum. Catalase decayed in these extracts. The addition of PMSF reduced the loss in all three of these activities. Fumarase was stable. The number of active fragments of isocitrate lyase recovered after filtration on Sephadex G-200 increased with the length of storage of crude extracts in the absence of PMSF at 4 C. Even in the presence of PMSF five activity peaks were observed after storage of extracts of C. elegans at 4 C for 72 hr. The molecular weights of active species ranged between 549,000 and 128,000 for isocitrate lyase in extracts of either C. elegans or A. suum. The 549,000- and 214,000-dalton species of isocitrate lyase from A. suum were much more labile at 50 C than the 543,000- and 195,000-dalton species from C. elegans.     

Enzymes of glucose metabolism in normal mouse pancreatic islets

1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP⁺-linked isocitrate dehydrogenase, `malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K<sub>m</sub> 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K<sub>m</sub> for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K<sub>m</sub> 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K<sub>m</sub> values of 2.5 and 21μm for NADP<sup>+</sup> and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K<sub>m</sub> values of 4 and 22μm for NADP<sup>+</sup> and glucose 6-phosphate. The K<sub>m</sub> for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP<sup>+</sup> and apparently mixed with respect to glucose 6-phosphate. 6. NADP<sup>+</sup>–isocitrate dehydrogenase was present but the islet homogenate contained little, if any, `malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the β-cell glucoreceptor mechanism.     

Initiation of citric acid accumulation in the early stages of Aspergillus niger growth

Summary Glycerol, which functions as an osmoregulator in the early stages of Aspergillus niger growth, slowly diffuses out of the cells and possibly into the mitochondria. Since mitochondrial nicotine adenine dinucleotide phosphate (NADP⁺)-specific isocitrate dehydrogenase is inhibited by glycerol, citrate starts to accumulate in the cells. At physiological pH values citric acid dissociates and affects the intracellular and extracellular pH. By the phosphorus-31-nuclear magnetic resonance technique a drop in intracellular pH from 7.1 to about 6.5 has been detected, which might significantly influence metabolic rates.