Donald Dudley Clarke PhD

Acetate and fluoroacetate as possible markers for glial metabolism in vivo.

[3H]Acetate has been shown by light autoradiographic methods to label the neuropil but not the perikarya in brain and retina. [3H]Fluoroacetate behaves similarly. The study provides anatomical data which support the concept of metabolic compartmentation of glutamic acid and associated metabolites previously derived from biochemical studies. It is suggested that these may be markers of non-neuronal metabolism, probably mostly glial, and may be used to develop procedures which will provide complementary data to that obtained with 2-deoxyglucose on regional metabolism in brain.

Fluoroacetate and fluorocitrate: Mechanism of action

The concept of lethal synthesis as suggested by Peters is reviewed in the light of the more recent work in this area. It is suggested that fluorocitrate is a “suicide” substrate for aconitase rather than a competitive inhibitor as originally suggested. The use of these substances to study glialneuronal relationships is considered.

Actomyosin-like protein isolated from mammalian brain.

A protein with characteristics similar to actomyosin has been isolated from whole brain of rat and cat. It is soluble in 0.6 molar potassium chloride and insoluble in 0.1 molar potassium chloride. It superprecipitates with magnesium ions and adenosine triphosphate. It has adenosine triphosphatase activity stimulated by either magnesium or calcium ions. Both superprecipitation and adenosine triphosphatase activity are inhibited by p-chloromercuribenzoate and Mersalyl but not by ouabain.

Determination of urinary 3-methoxy-4-hydroxyphenyl-ethylene glycol by gas-liquid chromatography and electron capture detection

The determination of urinary 3-methoxy-4-hydroxyphenylethylene glycol (HMPG), a major metabolite of epinephrine and norepinephrine by gas-liquid chromatography and electron capture detection is described. A purified extract of HMPG was treated with trifluoroacetic anhydride to form the highly sensitive HMPG-trifluoroacetate derivative. Chromatography was performed on a 6% QF-1-0065 column at 158°. Recovery was found to be 55.4% using [3H]MPG. Two sets of eight identical samples demonstrated a standard deviation from the mean of 6.9% and 5.7%. Thirty-five normal human urine specimens assayed by this procedure gave a mean HMPG excretion of o.86μg/mg creatinine (range 0.25–1.49). Six urine samples from patients with proven pheochromocytoma, and four from patients with proven neuroblastoma were analyzed and gave rise to the anticipated elevation in HMPG excretion. The high sensitivity of this procedure points to the possibility of estimation of plasma HMPG levels.

Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Glutamate dehydrogenase (GDH) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low‐speed pellet (480 g for 10 min). Approximately 60% of the high‐speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates μ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L‐leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 μM GTP for the soluble and particulate fractions, respectively. The HIll plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45°C and pH 7.4 in 50 mM phosphate buffer.

COMPARTMENTATION OF GLUTAMIC ACID METABOLISM IN BRAIN SLICES

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COMPARTMENTATION OF CITRIC ACID CYCLE METABOLISM IN BRAIN: EFFECT OF AMINOOXYACETIC ACID, OUABAIN AND Ca2+ ON THE LABELLING OF GLUTAMATE, GLUTAMINE, ASPARTATE AND GABA BY [1‐14C]ACETATE, [U‐14C]GLUTAMATE AND [U‐14C] ASPARTATE

—(1) The effects of aminooxyacetic acid, ouabain and Ca2+ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium‐[1‐14C]acetate, l‐[U‐14C]glutamate and l‐[U‐14C]aspartate as tracer metabolites.

COMPARTMENTATION OF CITRIC ACID CYCLE METABOLISM IN BRAIN: LABELLING OF GLUTAMATE, GLUTAMINE, ASPARTATE AND GABA BY SEVERAL RADIOACTIVE TRACER METABOLITES

—(1) Compartmentation of the metabolism of amino acids in brain has been studied in slices of cerebral cortex incubated with sodium [1‐14C]acetate, sodium [1‐14C]‐bicarbonate, [1‐14C]GABA or l‐[1‐14C]glutamate and in samples of brain after injection in vivo of [1‐14C]‐ or [3H]acetate.

Nerve tissue-specific human Glutamate dehydrogenase that is thermolabile and highly regulated by ADP

Abstract: Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH‐specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue‐specific GDH showed only 5% of its maximal activity, compared with ∼40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05–0.25 mM) induced a concentration‐dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550–1,300%) than of the housekeeping enzyme (by 120–150%). Magnesium chloride (1–2 mM) inhibited the nonactivated housekeeping GDH (by 45–64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue‐specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05–0.25 mM). Heat‐inactivation experiments revealed that the half‐life of the housekeeping and nerve tissue‐specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue‐specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.

A quantitative assay for vanillylmandelic acid (VMA) by gas-liquid chromatography

A quantitative assay for vanillylmandelic acid (VMA) by gas-liquid chromatography / Sherwin Wilk From the Department of Medicine, th eMount Sinai Hospital, New York, New York and the Department of Chemistry, Fordham University, New York, N.Y. Stanley E. Gitlow and Milton Mendlowitz From the Department of Medicine, The Mount Sinai Hospital, New York, New York Morton J. Franklin and Herman E. Carr From the Department of Psychiatry, Boston University, School of Medicine, Boston, Massachusetts and Donald D. Clarke From the Department of