An inhibitor of dopamine uptake,LR5182, CIS-3-(3,4-dichlorophenyl)-2-N,N-dimethylaminomethyl-bicyclo-[2,2,2]-octane,hydrochloride

LR5182 inhibited the uptake of dopamine in rat striatal synaptosomes and the uptake of norepinephrine in cortical synaptosomes with inhibitor constants, Ki values, of 3nM and 58nM, respectively. It was only a week inhibitor of serotonin uptake in cortical synaptosomes with a Ki value of 1.7μM. The uptake of dopamine and norepinephrine were significantly lowered within an hour after an intraperitoneal injection of LR5182. Among known inhibitors of dopamine uptake in synaptosomes of rat brain, LR5182 is most effective and selective. The rigid structure of LR5182 (Figure 1) suggested a gauche conformation of dopamine to be favored by the striatal uptake of dopamine.     

Characteristics of the Inhibition of Rat Brain Monoamine Oxidase In Vitro by MD780515

Abstract: The inhibition of type A and B MAO in rat forebrain crude membrane preparation by MD780515. (3-{4-[(3-cyanophenyl)methoxy]phenyl)-5-(methoxymethyl)-2-oxazolidinone Centre de Recherche Delalande, France) has been investigated in vitro with 5-hydroxytryptamine and β-phenylethyl-amine as substrates. The inhibition of the high-affinity binding of [³H]harmaline, a specific marker of type A MAO, was also studied. In the experimental conditions used, MD780515 appeared to be a pure mixed MAO inhibitor (MAOI) of 5-HT deamination, both K_m , and V_max being altered [K₁ (Dixon) = K_i , (slope) = 2 nM; K_i (intercept) = 12 nM]. Phenylethylamine oxidation could be considered to be noncompetitively inhibited by MD780515 (K_i (slope) = 78 nM; K_i , (intercept) = 103 nM). Dixon and intercept replots were hyperbolic, suggesting that, at high concentrations, PEA could be deaminated by both forms of MAO. This hypothesis was confirmed by biphasic inhibition curves of 80 μM-PEA obtained when MD7805 15 , clorgyline, harmaline and deprenyl were used as MAOIs. MD780515 was a potent inhibitor (IC₅₀= 1–2 nM) of [³H]harmaline binding. Comparatively, clorgyline, 'cold' harmaline and Lilly 51641 inhibited ³H ligand binding, with IC₅₀ of 5, 7 and 40 nM respectively. In conclusion, MD780515 is a reversible, specific and potent type A MAOI.     

CHARACTERISTICS OF D-GLUCOSAMINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

Abstract— The uptake of D-glucosamine by rat brain synaptosomes is studied as a function of time, temperature and synaptosomal protein and substrate concentrations. The rate of D-glucosamine uptake, after correcting for simple diffusion, obeys Michaelis-Menten kinetics. The apparent kinetic constants for the uptake process are K_m = 2.5 0.8 m m , V_max = 3.7 ± 1.2 nmol/mg protein/min. D-Glucose, D-mannose, 2-deoxy-D-glucose and 3-0-methyl-o-glucose are potent inhibitors of D-glucosamine uptake. 2-Deoxy-D-glucose and D-glucosamine inhibit the uptake of one another in a simple competitive manner, indicating their sharing of a common transport system. Cytochalasin B, phloretin and phloridzin are powerful competitive inhibitors of D-glucosamine uptake with apparent inhibitor constants ( K₁ ) of 7.0 × 10^-5, 2.3 × 10^-3 and 0.4 mM, respectively. The uptake is unaffected by Na⁺, Li⁺ and Mg²⁺, partially inhibited by NH₄⁺, Mn²⁺ and Ca²⁺, and slightly stimulated by PO₄-ions. D-Glucosamine uptake is also sensitive to inhibition by several sulfhydryl reagents, thus implying the involvement of sulfhydryl groups in the transport process. The apparent affinity constants for synaptosomal transport for both D-glucosamine and 2-deoxy-D-glucose are about 4 times greater in 7-day-old than in the adult rat brains.     

Blockade by Neurotransmitter Antagonists of Veratridine-Activated Ion Channels in Neuronal Cell Lines

Abstract: The voltage-dependent Na⁺ ionophore of various neuronal cells is permeable not only to Na⁺ ions but also to guanidinium ions. Therefore, the veratridine-(or aconitine-) stimulated influx of [¹⁴C]guanidinium in neuroblastoma × glioma hybrid cells was measured to characterize the Na⁺ ionophore of these cells. Half-maximal stimulation of guanidinium uptake was seen at 30 μ M veratridine. At 1 m M guanidinium, the veratridine-stimulated uptake of guanidinium was lowered to 50% by approximately 60 m M Li⁺, Na⁺, or K⁺ and by a few millimolar Mn²⁺, Co²⁺, or Ni²⁺. The basal, as well as the veratridine-stimulated, uptake of guanidinium was inhibited by the cholinergic antagonists (+)-tubocurarine ( K_i = 50 to 500 n M ) and atropine ( K_i = 5 to 30 μ M ) and the adrenergic antagonists phentolamine ( K_i = 5 μ M ) and propranolol ( K_i = 60 μ M ). The specificity of the inhibitory effects of these agents is stressed by the ineffectiveness of various other neurotransmitter antagonists. However, the corresponding ionophore in neuroblastoma cells (clone N1E-115) seems to be regulated differently. While phentolamine and propranolol inhibit the veratridine-activated uptake as in the hybrid cells, (+)-tubocurarine and atropine exert only a slight effect.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Anticholinesterase Action of a Bromine Compound Isolated from Human Cerebrospinal Fluid

Abstract: L-l-Methylheptyl-γ-bromoacetoacetate was found to be a competitive inhibitor of the acetylcholines-terases (electric eel, K_i= 17.2 μM; rat brain, K_i= 32.6 μM) and of butyrylcholinesterase (horse serum, K_i= 1.2 μM). The L-isomer was a more effective inhibitor than the D-isomer. The bromine atom at the γ-position of the acidic moiety, the specific length of the carbon chain constituting the secondary alcohol moiety, and the presence of the ketone radical at the acidic moiety of the ester were necessary for the anticholinesterase action. 1-Methyl-heptyl-γ-bromoacetoacetate formed a complex with acetylcholinesterase or butyrylcholinesterase without hydrolysis of its own molecule.     

Nicotine Indirectly Inhibits [3H]Dopamine Uptake at Concentrations That Do Not Directly Promote [3H]Dopamine Release in Rat Striatum

The effects of both (-)- and (+)-nicotine isomers were examined on in vitro uptake and release of [3H]dopamine in rat striatum. Both isomers inhibited uptake of [3H]dopamine in chopped tissue at concentrations well below those necessary for promoting release of preloaded [3H]dopamine. (-)-Nicotine was more potent than (+)-nicotine both at inhibiting uptake and at promoting release. Unlike other dopamine uptake inhibitors, however, nicotine inhibited only 50% of the total uptake. In the presence of 1 nM nicotine, the residual [3H]dopamine uptake was less sensitive to inhibition by cocaine than uptake in the absence of nicotine. Nicotine did not compete against the binding of [3H]GBR 12935, a selective dopamine uptake inhibitor. The nicotinic receptor agonists carbachol and 1,1-dimethyl-4-phenylpiperazinium iodide also inhibited uptake, whereas the nicotinic antagonists chlorisondamine and mecamylamine blocked nicotine's effect. Thus, the effect of nicotine on dopamine uptake appears to be mediated by a receptor similar to the nicotinic acetylcholine receptor. These receptors do not seem to be on the terminals that are accumulating dopamine, however, since tetrodotoxin prevented the effect of nicotine on [3H]dopamine uptake and nicotine had no effect on uptake in a synaptosomal preparation.     

A Synaptosomal Preparation from the Guinea Pig Ileum Myenteric Plexus

Abstract: Our interest in investigating the presynaptic modulation of acetylcholine release led to the development of a synaptosomal preparation from the guinea pig ileum myenteric plexus-longitudinal muscle. A crude synaptosomal fraction (P₂) was obtained by homogenization and differential centrifugation. The preparation exhibited a specific uptake system for choline and for nor-adrenaline (NA), but not for 5-hydroxytryptamine (5-HT). Synaptosomes were isolated from this P₂ fraction by an isoosmotic density gradient prepared from sucrose and metrizamide. The resultant synaptosomal fraction was enriched about sevenfold in both choline uptake and in choline acetyltransferase (ChAT). Choline was transported by a high-affinity system with a K_m of 6.5 × 10⁻⁷ M and a V_max of 41 pmol/mg protein/min. Electron microscopy confirmed the synaptosomal nature of the gradient fraction. Some synaptosomal profiles contained only small, translucent vesicles whereas others also contained large (approx. 100 nm diameter) electron-opaque vesicles. The crude synaptosomal fraction synthesized acetylcholine (ACh) from exogenous choline and it released the synthesized ACh in a calcium-dependent manner.     

Purification and properties of a neutral invertase from the roots of Cichorium intybus

Multiple activity peaks of neutral invertase (EC 3.2.1.26) were found in chicory roots ( Cichorium intybus L. var. foliosum cv. Flash). The main activity peak was purified by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, chromatofocusing and gel filtration. This protocol produced a 77-fold purification and a specific activity of 1.6 μmol (mg protein)⁻¹ min⁻¹. The mass of the enzyme was 260 kDa as estimated by gel filtration and 65 kDa on SDS-PAGE. Optimal activity was found between pH 7 and 7.5. The purified enzyme exhibited hyperbolic saturation kinetics with a K_m between 10 and 20 mM for sucrose. No other products than glucose and fructose could be detected. Raffinose was hydrolyzed at a rate of 2.4% relative to sucrose whereas the enzyme did not hydrolyze maltose, cellobiose, trehalose, 1-kestose, 1.1-nystose or inulin. Neutral invertase activity was completely inhibited by HgCl₂ and AgNO₃ and partially inhibited by CoCl₂, and ZnSO₄ (1 mM). Pyridoxal phosphate (K_i≅ 500 μ M ), Tris (K_i≅ 1.2 m M ), glucose and fructose (K_i≅ 16 m M ) were strong inhibitors of the enzyme. Fructose and Tris behaved as competitive inhibitors. A possible role for the enzyme's activity in vivo is discussed.     

MUSCIMOL UPTAKE, RELEASE AND BINDING IN RAT BRAIN SLICES

Abstract— The GABA analogue, muscimol, was taken up relatively inefficiently compared to GABA by slices of rat cerebral cortex at 37 C. Muscimol uptake followed saturation kinetics (K_m ImM. V_m 0.1 μmol g mini and showed an absolute dependence on sodium ions. The relative susceptibilities of muscimol uptake and GABA high affinity uptake to a variety of inhibitors, including (-)-nipecotic acid. (+)-2.4-diaminobutyric acid and arecaidine, and the stimulation of muscimol efflux by 50μM-GABA, suggest that muscimol and GABA share some common transport carriers. Since L-histidine inhibited muscimol uptake hut not GABA high affinity uptake, at least part of the observed muscimol uptake may be mediated by the 'small basic'amino acid transport system. Muscimol appeared to he taken up into nerve terminals, since uptake was inhibited by the neuronal uptake inhibitor cis -3-aminocyclohexanecarboxylic acid but not by the glial uptake inhibitor β-alanine. Muscimol efflux was stimulated in a calcium-dependent manner by an increased potassium ion concentration.
Sodium-independent binding of muscimol was observed in slices of rat cerebral cortex at 4 C. Binding could be inhibited by a variety of substances. including GABA, isoguvacine and (+)-bicuculline methochloride, which are known to inhibit the binding of muscimol to putative GABA receptors associated with synaptic membranes purified from rat brain.     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.     

Fucose Incorporation and Identification of Fucosylglycoproteins in Synaptosomes

Abstract— A synaptosome-enriched fraction from sheep cortex was incubated with l -fucose. The uptake of the sugar into this preparation was dependent on time, temperature, and concentration. A A K m_app of 0.94 m m - l -fucose and a V max_app value of 0.24 n m - l -fucose/mg synaptosomal soluble protein/20 min was determined. After incubation for 10 min at 25°C with l -[³H]fucose, 70% of the radioactive label was found in the soluble fraction. DEAE-cellulose chromatography resulted in the elution of three fucosylprotein peaks which were then characterised by gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). At least eleven ³H protein-staining bands were identified with M. W. 13,000-115,000. Control experiments involving the incubation of the hexose with heat-treated synaptosomes and myelin, mitochondria, and microsomes indicated that the tritiated material associated with the synaptosomal soluble fraction was not due to nonspecific binding or to the presence of contaminating subcellular material. A ³H glycopeptide was identified, and on analysis the carbohydrate moiety was found to be rich in sialic acid, fucose, galactose, mannose, and N -acetylglucosamine. Mild acid treatment of the glycopeptide released fucose, which implies that this carbohydrate occupies a terminal position in the oligosaccharide chain. From these results it is proposed that synthesis or the modification of soluble fucosylglycoproteins is possible in synaptosomes.     

SYNAPTOSOMAL PHOSPHORYLATION OF D-GLUCOSAMINE

Abstract— Synaptosomal fractions from rat brains can phosphorylate d -glucosamine once it is transported across the synaptosomal membrane. Intrasynaptosomal phosphorylation of d -glucosamine was rapid; up to 80% of the d -glucosamine was recovered as the phosphorylated amino sugar during 15 min of incubation with the substrate. Transport of d -glucosamine was the rate-limiting step in the uptake process since in the presence of cytochalasin B, a specific sugar transport inhibitor, the uptake of d -glucosamine was inhibited to the same extent as its phosphorylation. The pH optimum for phosphorylation of d -glucosamine by lysed synaptosomal preparations was between 7.5 and 9.0. The mean K _m and V _max values for this phosphorylation were 0.46 DIM and 676 nmol/mg protein per min, respectively. Phosphorylation of d -glucosamine was inhibited competitively by d -glucose, 2-deoxy- d -glucose and N -acetyl- d -glucosamine and uncompetitively by d -glucose-6-phosphate. The phosphorylation was strongly inhibited by several sulfhydryl reagents, but was insensitive to product inhibition, cytochalasin B, phloretin and phloridzin.     

γ-Glutamyl-transpeptidase in tobacc suspension cultures: Catalytic properties and subcellular localization

γ-Glutamyl-transpeptidase activity (EC 2.3.2.2) was found in ammonium sulfate precipitates of extracts from cultured cells of Nicotiana tabacum L. var. Samsun. Specific activity up to 3.2 nmol (mg protein)⁻¹ min⁻¹ was achieved, using the artificial substrate γ-glutamyl- p -nitroanilide (K_m 0.6 m M ) instead of glutathione. Optimal enzyme activity was obtained at pH 8.0–8.5 and 45°C. The enzyme reaction was inhibited competitively by γ-glutamyl analogs (6-diazo-5-oxo-L-norleucine: K_i 0.76 μ M ; L-azaserine: K_i 0.23 m M ) or the inorganic ion m -periodate (K_i 0.43 m M ). Cell fractionation and in vivo experiments revealed that 77% of the γ-glutamyl-transpeptidase activity is localized in the soluble cytoplasmic fraction, while 20–23% of the enzyme is found on the outer surface of the plasmalemma.     

Characterization of Receptors for Cholecystokinin and Related Peptides in Mouse Cerebral Cortex

Abstract: The characteristics of cholecystokinin (CCK) binding to its receptors in a particulate membrane fraction of mouse cerebral cortex were studied by employing biologically active radioiodinated CCK prepared by conjugation with ¹²⁵I-Bolton-Hunter (¹²⁵I-BH) reagent. At 24°C binding was rapid, reversible, and linearly related to protein content. Binding was maximal at acidic pH (6.5) and reduced by the presence of monovalent cations. Under physiological conditions (pH 7.4, 118 mM-NaC1, 4.7 mM-KCl) Scatchard plots of CCK binding were linear with a K _D value of 1.27 nM and binding capacity of 115 fmol/mg protein. Optimal binding required the presence of both Mg²⁺ and EGTA, and was inhibited by the addition of micromolar concentrations of Cu²⁺ (ID₅₀= 30 μM). The cortical receptor recognized all major forms of CCK, with an order of potency of cholecystokinin octapeptide (CCK₈) > CCK > cholecystokinin tetrapeptide (CCK₄). Desulfated cholecystokinin octapeptide (dCCK₈) had a 10-fold lower affhity than CCK₈. Dibutyryl cyclic GMP, a potent competitive inhibitor of CCK binding to receptors in pancreas, was not a specific inhibitor of CCK binding to brain receptors. These present results support the concept that CCK may function as a regulatory peptide in brain, and that the cortical CCK receptor is different from the receptors mediating the peripheral action of CCK.     

CHARACTERIZATION OF A DOPAMINE-SENSITIVE ADENYLATE CYCLASE IN THE RAT CAUDATE NUCLEUS

Abstract— An adenylate cyclase present in the caudate nucleus of rat brain, which is selectively stimulated by low concentrations of dopamine, and which is believed to mediate dopaminergic synaptic transmission, has been characterized with respect to several properties. The parameters studied included temperature, pH, ATP concentration, Mg/ATP ratio, and metal ion specificity. The effects of other compounds, including EGTA, NaF and several guanosine nucleotides, were also tested on the dopa-mine-sensitive adenylate cyclase. In addition, the subcellular distribution of the enzyme was studied. The highest specific activity was found in subcellular fractions enriched in nerve endings. A half-maximal increase in the activity of the enzyme in a subcellular fraction occurred in the presence of 4 × 10⁻⁶ M dopamine. Fluphenazine, a dopamine antagonist, competitively inhibited the activity of the enzyme in this fraction, with a calculated inhibition constant ( K_i ) of 8 × 10⁻⁹M.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Isolation and properties of β-glucan synthetase from the aquatic fungus, Aphanomyces astaci

A β-glucan synthetase was isolated from a membrane fraction of the crayfish parasitic fungus Aphanomyces astaci Schikora, strain Si. [¹⁴C]-UDP-glucose was incorporated linearly for about 1 h at 30°C into an acid insoluble product. The apparent K_m for UDP-glucose was found to be approximately 4.5 m M and the apparent K_i for UDP, a competitive inhibitor of the reaction, was 1 m M . The acid insoluble product obtained after incubating this glucan synthetase with[₁₄C]-UDP-glucose was partially characterized by glucanase treatment. This product mainly consisted of β-1,3-linked glucosyl units. Synthetase activity was not stimulated by MgCl₂, but cellobiose as well as GTP and EDTA in combination or ATP alone enhanced enzyme activity. A high proportion of the A. astaci synthetase was probably already activated during preparation and not accessible to further stimulation by nucleotide additions as found for glucan synthetase of Saccharomyces cerevisiae and Candida albicans. No synthetase activity or any factors affecting this enzyme was present in the cytosol. An exudate prepared from the cuticle of the crayfish host, did not inhibit glucan synthetase activity in vitro.     

Kinetic properties of phosphoenolpyruvate carboxylase from lupin nodules and roots

The kinetic properties of two forms of phosphoenolpyruvate carboxylase (PEPC I and PEPC II, EC 4.1, 1.31) from lupin ( Lupinus luteus L. cv. Ventus) nodules and one enzyme form (PEPC III) from roots were studied. The Michaelis constant (K_m) values for PEP, Mg²⁺ and especially HCO₃⁻were lower for PEPC I. Kinetic studies showed that aspartate is a competitive inhibitor at pH 7.2 and inhibitor constant (K_i) values are different for the three forms of PEPC. Malate is a competitive inhibitor for PEPC I and PEPC III and shows mixed-type inhibition for PEPC II. Malate inhibition is dependent upon the pH of the assay. Different effect of several metabolites was also observed. The temperature optimum was near 39°C for PEPC I and around 43°C for PEPC II and PEPC III. PEPC I appeared to be the most thermolabile. It is suggested that PEPC I from lupin nodules is closely associated with N₂ fixation.