Interconvertibility of molecular forms of 3'5' cyclic AMP phosphodiesterase of human platelets]

Three forms of cyclic AMP phosphodiesterase from human platelets are resolved upon sucrose gradient centrifugation : 2.5S (monomer), 4.8S (dimer) and 7S (tetramer). They are interconvertible and form an association-dissociation equilibrium depending on the concentration of enzyme. The dissociated form has a high Km for cyclic AMP (Km : 3-5.10(-4) M) whereas the associated form has a low Km (Km : 3-5.10(-5) M).     

3'5'-Cyclic AMP phosphodiesterase activity in the internal and external layers of human myometrium at the end of pregnancy]

The hydrolysis of cAMP by phosphodiesterase was studied in whole homogenate from human myometrium at the end of the pregnancy before onset of labour. Tissue samples were taken from outer and inner layers of placental and anti-placental sites. Kinetic analysis shows in every case two apparent Km values for low and high cAMP concentrations in the order of 1 x 10(-5) M and 1 x 10(-4) M. On the other hand Vmax values are lower for the enzyme isolated from the placental site than for the one isolated from the anti-placental area. In the 4 zones studied, an appreciable proportion of the low Km enzyme is present.     

Purification and properties of ornithine aminotransferase from rat brain

Ornithine aminotransferase (E.C. 2.6.1.13) from rat brain was purified 100-fold by ammonium sulphate fractionation, DEAE cellulose chromatography, calcium phosphate gel and alumina C gamma gel. Pyridoxal phosphate was essential for maximum activity of the enzyme. The brain enzyme did not differ from liver and kidney enzymes in properties such as pH optimum, Km, substrate specificity and the inhibition by branched chain amino acids. Unlike rat liver enzyme, brain ornithine aminotransferase was able to catalyze the reaction between L-lysine and 2-oxoglutarate. Spermidine and spermine inhibited brain ornithine aminotransferase activity.     

Allantoinase in the marine polychaete Eudistylia vancouveri.

Allantoinase, an enzyme in the purine-urea cycle, was found in Eudistylia vancouveri (Polychaeta). The enzyme had a pH optimum at 7.6. The Km was 0.012 M allantoin, and the Arrhenius energy of activation was 12.6 to 14.6 kcal/mol.     

Stimulation of the synthesis and non-competitive inhibition of alkaline phosphatase by theophylline in normal hamster fibroblasts and absence of response in transformed fibroblasts]

Theophylline increases the synthesis of alkaline phosphatase in normal Hamster fibroblasts (activity X6 after 24 hours with theophylline 10-3 M). The enzymatic activity of these cells is inhibited by theophylline in vitro (80% inhibition in the presence of theophylline 10-3 M). The inhibition seems to be non competitive, since the apparent Km of the enzyme is not modified. This stimulator and inhibitor effect of theophylline is absent in Hamster fibroblasts transformed by SV 40 virus.     

S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localisation

S-nitrosoglutathione (GSNO) formation represents a mechanism for storage and transport of nitric oxide. Analysis of human liver and Saccharomyces cerevisiae extracts has revealed the presence of only one enzyme able to significantly reduce GSNO, identified as glutathione-dependent formaldehyde dehydrogenase (FALDH). GSNO is the best substrate known for the human and yeast enzymes (kcat/Km = 444,400 and 350,000 mM(-1) min(-1), respectively). Although NADH is the preferred cofactor, some activity with NADPH (Km = 460 microM) can be predicted in vivo. The subcellular localization demonstrates a cytosolic and nuclear distribution of FALDH in living yeast cells. This agrees with previous results in rat, and suggests a role in the regulation of GSNO levels in the cytoplasmic and nuclear compartments of the eukaryotic cell.     

Effect of phosphonic analogues of glutamic acid on glutamate decarboxylase

Among the phosphonic analogues of glutamic acid, only 4-amino-4-phosphono butyric acid, the compound which shows the highest affinity for pyridoxal phosphate, inhibits competitively both Escherichia coli and rat brain glutamate decarboxylases. Phosphinothricin, 2-amino-4-(methylphosphino)butyric acid, is a strong inhibitor of the mammalian enzyme.     

Platelet monoamine oxidase B: use and misuse

The human platelet in addition to having serotonin (5-HT) receptors, uptake carriers (receptor) and transmitter storage vesicles, primarily possesses mitochondrial monoamine oxidase (MAO) type B. Similar to the major form of MAO in the human brain, this enzyme actively oxidizes A-B and B substrates (tyramine, dopamine, phenylethylamine) as well as the novel secondary amine anticonvulsant, milacemide and dopaminergic neurotoxin, MPTP. 5-HT oxidation is hardly affected by the platelet enzyme and MAO inhibitors have no net effect on its accumulation. MAO-B is selectively inhibited by 1-deprenyl and thus the platelet enzyme may be useful to monitor the anti-Parkinson activity of such drugs, as related to their ability to inhibit brain MAO-B. The oxidation of the anticonvulsant, milacemide, to glycine in vitro and in vivo by MAO-B, may herald new prospects for the development of inert prodrugs capable of being metabolized to neuroactive substances by MAO-B. The plasma levels of their metabolites may be an index of MAO-B activity found in the platelet and brain.     

Platelet monoamine oxidase B: Use and misuse

The human platelet in addition to having serotonin (5-HT) receptors, uptake carriers (receptor) and transmitter storage vesicles, primarily possesses mitochondrial monoamine oxidase (MAO) type B. Similar to the major form of MAO in the human brain, this enzyme actively oxidizes A-B and B substrates (tyramine, dopamine, phenylethylamine) as well as the novel secondary amine anticonvulsant, milacemide and dopaminergic neurotoxin, MPTP. 5-HT oxidation is hardly affected by the platelet enzyme and MAO inhibitors have no net effect on its accumulation. MAO-B is selectively inhibited by 1-deprenyl and thus the platelet enzyme may be useful to monitor the anti-Parkinson activity of such drugs, as related to their ability to inhibit brain MAO-B. The oxidation of the anticonvulsant, milacemide, to glycine in vitro and in vivo by MAO-B, may herald new prospects for the development of inert prodrugs capable of being metabolized to neuroactive substances by MAO-B. The plasma levels of their metabolites may be an index of MAO-B activity found in the platelet and brain.     

Effect of phosphonic analogues of glutamic acid on glutamate decarboxylase

Summary Among the phosphonic analogues of glutamic acid, only 4-amino-4-phosphono butyric acid, the compound which shows the highest affinity for pyridoxal phosphate, inhibits competitively bothEscherichia coli and rat brain glutamate decarboxylases. Phosphinothricin, 2-amino-4-(methylphosphino)butyric acid, is a strong inhibitor of the mammalian enzyme.We acknowledge the gift of a sample of phosphinothricin by Prof. Przemyslaw Mastalerz as well as the technical assistance of Denois.     

Cleavage of des-Arg9-bradykinin by angiotensin I-converting enzyme from pig kidney cortex

Fast and very slow hydrolyses of des-Arg9-bradykinin and angiotensin II by angiotensin I-converting enzyme were detected by high performance liquid chromatography. The Michaelis constants of the enzyme, Km values, for des-Arg9-bradykinin and bradykinin were found to be 0.24 mM and 4.4 microM, and the maximum velocities, Vmax values (mumol . min-1 . mg protein-1) for these compounds to be 3.24 and 0.34, respectively. The enzyme also hydrolyzed Z-Gly-Pro-Gly-Gly-Pro-Ala to a tripeptide that was identified as dansyl-Gly-Pro-Ala by TLC on polyamide. These observations show that the enzyme hydrolyzes the peptides at the bond before the prolyl residue in the penultimate position.     

Purification of calf thymus adenosine diphosphate ribose polymerase]

Calf thymus poly ADPR polymerase has been purified to electrophoretic homogenity. The enzyme has a molecular weight of 120,000 +/- 10,000 dalton. The substrate affinity is very high (apparent Km 82.5 micrometer). The presence of exogenous DNA does not appear to be a requisite for enzymatic activity of the purified enzyme.     

Oxidation of synephrine by type A and type B monoamine oxidase.

Synephrine (SP) was found to be a substrate for monoamine oxidase (MAO) in rat brain mitochondria, showing the Km and Vmax values of 250 microM and 32.6 nmoles/mg of protein/30 min respectively. The inhibition studies showed that the SP oxidation was carried out by both type A and type B MAO and a major part of the activity was due to type A MAO.     

Use of a zonal rotor for demonstrating that human myelin is formed by a continuum of particles of different densities]

Human myelin isolated from frozen brain consists of a continuum of particles as shown by sucrose continuous gradient (between 0,4 and 0,85 M) on zonal rotor. This was determined by measuring the O. D. at 260 and 280 millimicron, the protein concentration by Lowry method and sucrose concentration by polarimetry in 60 fractions. Most of the material is concentrated between 20-23% sucrose, the maximum being found at 21.5% (0.63 M). The fractions are dialyzed for 62 hrs. to remove the sucrose and the pellet obtained after centrifugation shows that the protein concentration increases from the ligher fraction to the heavier.     

Calcium dependence of rat brain tryptophan hydroxylase.

The effects of various concentrations of ionized Ca were examined on the activity of rat brain tryptophan hydroxylase previously treated with EGTA. Within the range of ionized Ca-concentrations though to be physiologically important (10(-8) to 10(-5) M), no significant activation of the enzyme occurred, although activation was observed at higher concentrations of the metal.     

Gamma-aminobutyric acid uptake by rat kidney brush-border membrane vesicles

Summary Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for -aminobutyric acid (GABA), a high affinity system (Km=10.9 M) and a low affinity system (Km=1203 M). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, -alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMW uptake systems were clearly different from the GABA transport systems present in brain tissue.     

gamma-Aminobutyric acid uptake by rat kidney brush-border membrane vesicles

Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for gamma-aminobutyric acid (GABA), a high affinity system (Km = 10.9 microM) and a low affinity system (Km = 1203 microM). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, beta-alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5 c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMV uptake systems were clearly different from the GABA transport systems present in brain tissue.     

A convulsant, 3-mercaptopropionic acid,decreases the level of GABA and GAD in rat pancreatic islets and brain

To investigate the properties of the gamma-aminobutyric acid (GABA) synthesizing enzyme, glutamate decarboxylase (GAD), in the brain and the pancreatic islets of the rat, GABA concentration in the brain and the pancreatic islets was measured after intraperitoneal administration of 3-mercaptopropionic acid (3-MP) at 25 mg/kg. 60 min after the administration of 3-MP, GABA concentration in the hypothalamus, the superior colliculus and the hippocampus of the brain decreased by 20–30% and in the pancreatic islets by 35%. The concentration in the pancreatic acini did not change. Western blotting showed that GAD activity in the pancreatic islets decreased after administration of 3-MP compared to the control. The activity of GAD in the pancreatic islets as well as brain can be modified by a convulsant, in this case 3-MP. These results suggest the properties of GAD may be similar in the pancreatic islets and brain.     

Nature of inhibition of rat testicular alkaline phosphatase by isatin

Isatin has been found to inhibit rat testicular alkaline phosphatase (EC 3.1.3.1). That the inhibition is non-competitive as well as non-allosteric is evident from a) the hyperbolic curve relating inhibition as a function of inhibitor concentration; b) the small change in enthalpy, free energy and entropy; c) the number of isatin molecules associating with 1 molecule of the enzyme (n=1.29); and, d) the decrease in the values of both Km and Vmax in the presence of isatin.     

Involvement of extrasynaptic glutamate in physiological and pathophysiological changes of neuronal excitability

Glutamate is the most abundant neurotransmitter of the central nervous system, as the majority of neurons use glutamate as neurotransmitter. It is also well known that this neurotransmitter is not restricted to synaptic clefts, but found in the extrasynaptic regions as ambient glutamate. Extrasynaptic glutamate originates from spillover of synaptic release, as well as from astrocytes and microglia. Its concentration is magnitudes lower than in the synaptic cleft, but receptors responding to it have higher affinity for it. Extrasynaptic glutamate receptors can be found in neuronal somatodendritic location, on astroglia, oligodendrocytes or microglia. Activation of them leads to changes of neuronal excitability with different amplitude and kinetics. Extrasynaptic glutamate is taken up by neurons and astrocytes mostly via EAAT transporters, and astrocytes, in turn metabolize it to glutamine. Extrasynaptic glutamate is involved in several physiological phenomena of the central nervous system. It regulates neuronal excitability and synaptic strength by involving astroglia; contributing to learning and memory formation, neurosecretory and neuromodulatory mechanisms, as well as sleep homeostasis.The extrasynaptic glutamatergic system is affected in several brain pathologies related to excitotoxicity, neurodegeneration or neuroinflammation. Being present in dementias, neurodegenerative and neuropsychiatric diseases or tumor invasion in a seemingly uniform way, the system possibly provides a common component of their pathogenesis. Although parts of the system are extensively discussed by several recent reviews, in this review I attempt to summarize physiological actions of the extrasynaptic glutamate on neuronal excitability and provide a brief insight to its pathology for basic understanding of the topic.