Christopher D. Cash

Comparison of the structural characteristics of the 4-aminobutyrate:2-oxoglutarate transaminases from rat and human brain, and of their affinities for certain inhibitors.

4-Aminobutyrate:2-oxoglutarate (4-aminobutyrate:2-oxoglutarate amino-transferase, EC 2.6.1.19) from human brain has been purified 2500-fold with respect to the initial homogenate. The enzyme, which appears to be pure by polyacrylamide gel electrophoresis, N-terminal analysis and immunodiffusion, was compared to rat brain 4-aminobutyrate transaminase, purified to the same extent in an earlier study [15]. The two enzymes, which have approximately the same molecular weight, show large differences in their tryptic fingerprints and in the peptides produced by cyanogen bromide cleavage. The Km values (limit) for 4-aminobutyrate are different, the human enzyme having four times greater affinity for this substrate. A series of branched-chain fatty acids (including n-dipropylacetate), which are structural analogues of 4-aminobutyrate and inhibit rat brain 4-aminobutyrate transaminase, are less powerful inhibitors of the human enzyme.

Isolation of a rat pineal gland cDNA clone homologous to tyrosine and phenylalanine hydroxylases

A rat pineal gland cDNA expression library has been probed with an antiserum raised against rat tryptophan hydroxylase. A clone has been isolated and its sequence reveals a high degree of homology with those of tyrosine and phenylalanine hydroxylases.

γ-hydroxybutyrate receptor function studied by the modulation of nitric oxide synthase activity in rat frontal cortex punches

Previous results have shown that stimulation of the gamma-hydroxybutyrate (GHB) receptor modulates Ca2+ channel permeability in cell cultures. In order to confirm this result, we investigated the consequence of GHB receptor stimulation on nitric oxide synthase (NOS) activity in rat brain cortical punches rich in GHB receptors. The stimulation of these receptors by increasing amounts of GHB induced a progressive decrease in NOS activity. However, for GHB doses above 10 microM, this reduction was progressively lost, either after receptor desensitization or after stimulation of an additional class of GHB receptor having lower affinity. The effect of GHB was reproduced by the GHB receptor agonist NCS-356 and blocked by the GHB receptor antagonist NCS-382. The GHB-induced effect on Ca2+ movement was additive to those produced by veratrine, indicating that GHB modulates a specific Ca2+ conductance, which explains the modification in NOS activity and the increase in cyclic guanosine monophosphate levels previously reported.

Purification and properties of two succinate semialdehyde dehydrogenases from human brain

In human brain there are two major isoenzymes of succinate semialdehyde dehydrogenase (succinate-semialdehyde: NAD+ oxidoreductase, EC 1.2.1.24). They are composed of two apparently identical subunits with a molecular weight of 69 000. The Km (limits) for their substrates NAD+ and succinate semialdehyde are 1.6.10(-5) M and 3.7.10(-6) M, respectively, for one enzyme, and 1.85.10(-5) M and 2.10(-6) M, respectively, for the other. These values, and other kinetic data obtained from the two enzymes are not very different. However the enzymes differ in the following respects: their behavior on ion exchange and 5-AMP affinity columns, their isoelectric points, their tryptic fingerprints and in their amino acid compositions.

Purification and properties of rat brain succinic semialdehyde dehydrogenase.

Succinic semialdehyde dehydrogenase from rat brain has been purified to electrophoretic homogeneity. It has a molecular weight of about 140, 000 and is composed of two apparently identical subunits. The reaction catalized by the pure protein is entirely dependent on endogenous --SH groups. The Kim (limits) for NAD and succinic semialdehyde are 2 X 10(-5) M and 1 X 10(-4) M respectively at the optimum pH of 8.6. Inhibition studies show that the reaction mechanism is a compulsory ordered on where NAD binds first followed by succinic semialdehyde.

Purification and partial characterisation of 4-aminobutyrate 2-ketoglutarate transaminase from human brain

A parallelism has been shown between the anticonvulsive effect and an increase in the cerebral GABA level after in vivo administration of some branched chain fatty acids such as n-dipropylacetate [ 1,2] . It has also been shown that these compounds improve conditioned behaviour [3] . It was later demonstrated that these compounds are competitive inhibitors of rat and mouse cerebal GABA-T**, thus probably explaining these effects [4]. This has stimulated interest in the search for non toxic compounds, slowly metabolised by the body, yet being powerful inhibitors of cerebral GABA-T. In order to test these compounds with human therapy in mind, it seemed pertinent to use GABA-T from human brain. We thus proceeded to purify this enzyme guided by the methods we employed for purification of GABA-T from rat brain [ 51. This human GABA-T preparation will also enable a comparison to be made with brain GABA-T from other species, in particular that of the mouse [6] and that of the rat [5] .

Ontogeny and distribution of specific succinic semialdehyde reductase apoenzyme in the rat brain

The ontogeny and distribution in rat brain of specific succinic semialdehyde reductase is described. This enzyme is probably responsible for the synthesis of γ-hydroxybutyrate in brain. The highest activities and levels of apoenzyme are found in cerebellum, olfactory bulb, septum and median hypothalamus. During neonatal development, the enzyme activity remains stable at least until 63 days of age. As the levels of other enzymes of the GABA shunt pathway increase during this same period, this result indicates that there is a relative decrease in the reductive pathway of succinic semialdehyde catabolism during development leading to γ-hydroxybutyrate synthesis, compared to the oxidative pathway leading to succinate.

Specific immunolysis of serotonergic nerve terminals using an antiserum against tryptophan hydroxylase

An antiserum to tryptophan hydroxylase purified from whole rat brain when incubated with rat striatal synaptosomes in the presence of complement caused release of 18% of LDH, 20% loss of potassium and 60% loss of tryptophan hydroxylase. Uptake of 5‐HT was reduced by 60%. Anti‐tryptophan hydroxylase alone, or complement alone were without action. The antiserum plus complement had no effect on DA uptake and did not release TH or GAD. These results suggest selective lysis of serotonergic nerve terminals had occurred. The antiserum plus complement reduced choline uptake by 45%. However, this did not seem due to lysis of cholinergic terminals, as ChAT was not released.

The immunolysis, isolation, and properties of subpopulations of mammalian brain synaptosomes

Five subpopulations of mammalian brain synaptosomes can be selectively damaged by complement-mediated immunolysis employing antibodies to specific surface markers for each subpopulation. This allows the size of these subpopulations to be estimated. Employing antibodies alone, it has proved possible to isolate three of these subpopulations in very pure preparations which are metabolically viable. The immunoaffinity technique involved (immunomagnetophoresis) uses magnetic microspheres and produces mg (protein) quantities of synaptosomes.

Isolation of Monoaminergic Synaptosomes from Rat Brain by Immunomagnetophoresis

Abstract: Monoaminergic synaptosomes have been isolated and purified from rat brain by immunomagnetophoresis. This novel technique uses magnetic beads to which Protein A is bound. Noradrenergic, dopaminergic, and serotonergic synaptosomes (previously cell‐surface labelled with anti‐dopamine‐β‐hydroxylase, anti‐tyrosine hydroxylase, and anti‐tryptophan hydroxylase, respectively) may be isolated in a highly purified state. The synaptosomal subpopulations are recovered in a viable metabolic state and show glucose‐stimulated respiration and Ca2+‐dependent neurotransmitter release. A novel subtype of dopamine‐β‐hydroxylase was found in dopaminergic terminals. No evidence for glutamate core‐lease from monoaminergic synaptosomes was obtained.