Acetylcholine Synthesis in the Schwann Cell and Axon in the Giant Nerve Fiber of the Squid

Abstract: Acetyltransferase enzymatic activity was detected and measured in homogenates obtained from intact nerve fibers and their separate cellular components, in the tropical squid Sepioteuthis sepioidea. The levels of acetylcholine synthesis were determined in pooled samples of whole stellar nerve, intact giant nerve fiber, extruded axoplasm, axoplasm-free giant nerve fiber sheaths, and small nerve fibers. The values found per mg of protein for the axoplasm-free sheaths are about 3–9 times those of the extruded axoplasm, and comparable to those found for the intact giant nerve fiber. These experimental findings settle the question of whether the Schwann cells of the giant nerve fiber of S. sepioidea , under physiological conditions, contain acetyltransferase activity and are able to synthesize acetylcholine.     

Aspartate Aminotransferase Activity in Fiber Tracts of the Rat Brain

Activity of aspartate aminotransferase, an enzyme which catalyzes the interconversion of the excitatory transmitter candidates, glutamate and aspartate, has been measured in fiber tracts of rat, with an emphasis on sensory and motor systems of the brain. Most tracts had significantly higher activities than the cholinergic facial nerve root, consistent with the possibility that a component of aspartate aminotransferase activity might serve as a marker for neurons using glutamate and/or aspartate as neurotransmitter. Highest activity was in the auditory nerve root. On the other hand, a close correlation was found between aspartate aminotransferase and malate dehydrogenase activities in the fiber tracts, raising the question whether aspartate aminotransferase activity may be more closely related to energy metabolism than to transmitter metabolism.     

ACETYLCHOLINE CONTENT OF THE SCHWANN CELL AND AXON IN THE GIANT NERVE FIBRE OF THE SQUID

Abstract— Acetylcholine and choline were identified and their concentrations measured, by means of gas chromatography/mass spectrometry, in extracts obtained from nerve fibers of the hindmost stellar nerve of the squid Sepioteuthis sepioidea. These compounds were quantitated in samples of stellar nerve devoid of giant fiber, intact giant nerve fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. In 11 samples of stellar nerve devoid of giant fiber, weighing an average of 20.8 ± 2.3 mg ( s.e.m. ), 756 ± 91 pmol ACh and 8.65 ± 0.62 nmol of choline were found. The total ACh content of the largest fibre in this group (10 μ m in diameter), for a 5 cm length of nerve, is in the order of 0.16 pmol. The average wet weights of a single giant nerve fiber (270-420 μ m in diameter) and its separate components ( s.e.m .; in mg; number of fibers in parentheses) were: intact fiber, 4.58 ± 0.19 (25); extruded axoplasm, 3.38 ± 0.13 (20); sheaths, 1.21 ± 0.11 (16). The average ACh content per unit weight of sample was about 2-3 times higher in the sheaths (5-13 pmol-mg⁻¹) than in the axoplasm (2-4 pmol mg⁻¹), whereas the ACh concentrations estimated per unit volume of cellular water were about 40 times higher in the Schwann cell (107-222 μ m ) than in the axon (2-5 μ m ). These experimental findings establish the presence of ACh in the giant nerve fiber of S. sepioidea. They also indicate the Schwann cells themselves as the main source for the release of ACh, responsible for their long-lasting hyperpolarizations following the conduction of nerve impulse trains by the axon.     

Glutamate Uptake by Squid Nerve Fiber Sheaths

Abstract: The squid giant nerve exhibits neuron-Schwann cell interactions that appear to involve glutamate as a mediator; however, there is no information available about the possible fate of the released glutamate. In this study, it is demonstrated that the periaxonal sheaths of the extrasynaptic regions of squid giant nerves (where the glial cells are located) possess the capacity to transport glutamate. In whole intact nerves incubated with low-glutamate concentrations for long periods of time, the majority of the glutamate incorporated into the tissue was found in the sheaths. Axoplasm-free sheaths incubated for long periods of time with low concentrations of glutamate were able to accumulate this amino acid against a large apparent concentration gradient. Sheath glutamate uptake occurred in a sodium-dependent fashion over a wide concentration range and displayed both high- and low-affinity components. Glutamate uptake at concentrations below the K _m of the high-affinity component was independent of homoexchange and displayed a specificity that is similar to that described for high-affinity glutamate transport in mammalian brain. It is proposed that the sheath transport systems may be involved in the regulation of glutamate levels in the intercellular clefts of the nerve fiber, as part of the glutamatergic neuron-glial signaling mechanisms in the squid giant nerve fiber.     

Distribution of some enzymes associated with the metabolism of glutamate, aspartate, gamma-aminobutyrate and glutamine in cat spinal cord

The activities of glutamine synthetase, glutaminase, glutamate decarboxylase, GABA aminotransferase, glutamate dehydrogenase, and aspartate aminotransferase were measured in four areas of the cat spinal cord and in dorsal and ventral roots. Five of the six enzymes showed identical distribution patterns; i.e. the activities in the dorsal and ventral gray matter were equal and those of dorsal and ventral white matter were equal. No statistical differences in the mean enzyme activities in the dorsal and ventral roots were found. Glutamate decarboxylase was the only enzyme which had a different pattern. The enzyme activity in dorsal gray was twice that of ventral gray; the same pattern as the GABA concentration in both these areas. The glutamine synthetase activities in the cord areas and roots correlated with the glutamine distribution reported earlier. Thus, the distribution of glutamine (not a transmitter) and GABA (questionable transmitter) in gray matter are dictated by their synthesizing enzymes, whereas the distribution of glutamate and aspartate (likely transmitter suspects) cannot be explained on the basis of enzyme activities. Therefore, the enzyme activities may be related to the amino acid levels primarily in metabolic compartments, whereas the excess of certain amino acids in specific areas of the cord and roots may be related to functional compartments accumulated for use in synaptic transmission.     

Alanine aminotransferase and some other enzymes in different populations of free brain cortex mitochondria

The activity of certain key enzymes involved in glutamic acid metabolism was studied in purified brain mitochondria and in mitochondrial subfractions separated in a discontinuous 1.2--1.6 mol/l sucrose gradient. Alanine aminotransferase and glutamate dehydrogenase were found to be matrix enzymes and aspartate aminotransferase to be associated with the inner mitochondrial membranes. After the purified mitochondria had been separated into 5 subfractions, aspartate aminotransferase and NAD+-dependent isocitrate dehydrogenase were found to be bound to the lighter mitochondrial subfractions settling at the 1.4--1.5 mol/l sucrose boundary while alanine aminotransferase, 4-aminobutyrate transaminase and glutamate dehydrogenase were associated with the heavier subfractions settling below 2.4 mol/l sucrose. The highest specific activity of the given enzymes was found in the subfraction settling at the 1.4--1.5 mol/l sucrose boundary, the only exception being alanine aminotransferase activity, whose maximum was found in the subfractions settling in 1.5 and 1.6 mol/l sucrose. It was concluded that alanine aminotransferase, in conjunction with glutamate dehydrogenase, is linked to NH3 binding and to the oxidation of reduced adenine nucleotides; in addition, alanine aminotransferase is presumed to have the function of transporting glutamate from the mitochondria to the extramitochondrial space.     

Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.

The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.

Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Changes in the activities of enzymes involved in amino acid metabolism during the senescence of detached wheat leaves

The activities of several enzymes related to amino acid metabolism were investigated in senescing detached wheat leaves ( Triticum aestivum L. cv. Diplomat) in light and darkness and after kinetin treatment. Glutamine synthetase and glutamate synthase activities rapidly declined in darkness. In light, the decline of glutamate synthase activity was retarded, while the activity of glutamine synthetase remained high and even increased transitorily. Kinetin treatment counteracted the decline of the activities of both enzymes. The activity of glutamate dehydrogenase markedly increased during senescence, particularly in light, and kinetin treatment lowered its activity. The activities of glutamate-oxaloacetate and glutamate-pyruvate amino-transferases and of NADP-dependent isocitrate dehydrogenase also increased in detached wheat leaves in light. Kinetin treatment prevented the rise of these enzyme activities. In darkness, the activities of glutamate-oxaloacetate aminotransferase and NADP-dependent isocitrate dehydrogenase decreased slowly while the decline of glutamate-pyruvate aminotransferase activity was more rapid. The activity of NAD-dependent malate dehydrogenase decreased both in light and, more rapidly, in darkness. The pattern of changes of the enzyme activities provides an explanation for the amino acid transformations and the flow of amino nitrogen into transport metabolites in senescing leaves.     

NEURONAL-GLIAL CONTRIBUTIONS TO TRANSMITTER AMINO ACID METABOLISM: STUDIES WITH KAINIC ACID-INDUCED LESIONS OF RAT STRIATUM

Abstract– Various aspects of amino acid metabolism were studied in striatum of rats with unilateral, kainic acid-induced lesions. Tissue slices were prepared from the lesioned and the contralateral, unlesioned, striatum. The preparations were incubated with a mixture of d -[2-¹⁴C]glucose and [³H]acetate in a Krebs-Ringer bicarbonate medium to evaluate oxidative metabolism. Glutamate and aspartate levels were decreased in the slices prepared from the lesioned striata by 35-40% and that of GABA by 75% compared to the levels found in the slices from the contralateral striata; glutamine levels were not different in the two preparations. Glucose utilization was decreased 60% in the slices from the lesioned striatum; this was caused not only by decreased levels of glutamate, aspartate and GABA but also by a decreased rate of labelling of glutamate and aspartate. On the other hand, the metabolism of [³H]acetate was greatly increased. The specific activities of glutamate and aspartate were 4-5-fold higher in the slices from kainic acid-lesioned striata; those of glutamine and GABA were unchanged. Thus, there was a 6-7-fold increase in the ratio of ³H to ¹⁴C in the specific activities of glutamate, aspartate and GABA with no change in this ratio in glutamine. The labelling of glutamine relative to that of glutamate, especially from [³H]acetate, suggested that the compartmentation of the glutamate-glutamine system was greatly altered in the kainate-lesioned striatum which now more closely resembled a single compartment system. The activities of lactate dehydrogenase, glutamate dehydrogenase, GABA transaminase and ‘cytoplasmic’ aspartate aminotransferase were decreased in homogenates of lesioned striatum. Succinate dehydrogenase, glutaminase (phosphate-activated) and ‘mitochondrial’ aspartate aminotransferase activities were unchanged whilst that of glutamine synthetase was increased. The results are consistent with hypotheses concerning the assignment of labelled acetate metabolism to glial cells as well as the distribution of the above enzymes between glia, neurones and nerve endings.     

The subcellular distribution of rat liver l-alanine–glyoxylate aminotransferase in relation to a pathway for glucose formation involving glyoxylate

1. The distribution of l-alanine-glyoxylate aminotransferase activity between subcellular fractions prepared from rat liver homogenates was investigated. The greater part of the homogenate activity (about 80%) was recovered in the ;total-particles' fraction sedimented by high-speed centrifugation and the remainder in the cytosol fraction. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients revealed a specific association between the aminotransferase and the mitochondrial enzymes glutamate dehydrogenase and rhodanese. 3. The aminotransferase activities in the cytosol and the mitochondria are due to isoenzymes. The solubilized mitochondrial enzyme has a pH optimum of 8.6, an apparent K(m) of 0.24mm with respect to glyoxylate and is inhibited by glyoxylate at concentrations above 5mm. The cytosol aminotransferase shows no distinct pH optimum (over the range 7.0-9.0) and has an apparent K(m) of 1.11mm with respect to glyoxylate; there is no evidence of inhibition by glyoxylate. 4. The mitochondrial location of the bulk of the rat liver l-alanine-glyoxylate aminotransferase activity is discussed in relation to a pathway for gluconeogenesis involving glyoxylate.     

The relationship between mitochondrial heterogeneity and gluconeogenesis in liver mitochondria of the rat, pigeon and guinea pig.

Isolated mitochondria of pigeon and guinea pig liver were subjected to zonal centrifugation. With pigeon liver mitochondria there was uniform distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, aspartate aminotransferase and glutamate dehydrogenase activities. Guinea pig liver mitochondria demonstrated two pyruvate carboxylase and phosphoenolpyruvate carboxykinase maxima but only one maximum with aspartate aminotransferase, malate dehydrogenase and glutamate dehydrogenase. Mitochondrial enzyme levels in rat, pigeon and guinea pig indicate different roles of certain gluconeogenic enzymes in the transport of carbon and hydrogen in and out of mitochondria.     

Pentylenetetrazole inhibits glutamate dehydrogenase and aspartate aminotransferase, and stimulates GABA aminotransferase in homogenates from rat cerebral cortex

The mechanism by which pentylenetetrazole provokes convulsions in animals has been investigated by measuring its influence in vitro on the activities of several enzymes of glutamate metabolism in rat brain homogenates. Pentylenetetrazole does not affect the specific activities of glutamine synthetase, glutaminase, or glutamate decarboxylase; it inhibits those of glutamate dehydrogenase and aspartate aminotransferase, and stimulates that of gamma-aminobutyric acid (GABA) aminotransferase. The overall consequence of the action of pentylenetetrazole on the activities of these enzymes should be an increase in the concentration of glutamate and a decrease in that of GABA. This modulation of glutamate and GABA metabolism by pentylenetetrazole could contribute to the triggering of convulsions.     

Effects of denervation on the activities of some tricarboxylic acid-cycle-associated dehydrogenases and adenine-metabolizing enzymes in rat diaphragm muscle

1. The activity of several tricarboxylic acid-cycle-associated dehydrogenases, adenine-metabolizing enzymes and glutathione reductase and the content of myoglobin were measured in rat diaphragm muscle after unilateral nerve section. 2. Consistent with morphological disintegration of the mitochondria there was a rapid diminution in activity of NAD- and NADP-linked isocitrate dehydrogenase, malate dehydrogenase and glutamate dehydrogenase. 3. Creatine phosphokinase and adenylate kinase, by contrast, showed little change in activity; adenylate deaminase and glutathione reductase activities increased during the hypertrophic phase. The concentration of myoglobin at first declined, then increased again. 4. The distribution of enzymes between the left and right hemidiaphragms was found not to be uniform. 5. Activities of adenine-metabolizing enzymes in the diaphragm were as great as in white muscle. It is suggested that their reputedly lower activities in red muscle properly refer to muscle containing a high proportion of intermediate fibres, which is not the case with diaphragm. 6. The possible causes of the transient hypertrophy after nerve section are discussed.     

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium.

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and L-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.     

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by (13)C and (31)P nuclear magnetic resonance

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.     

Cell growth in arginine- and ornithine-deprived medium and conversion of glutamate to ornithine and arginine in rat hepatoma cells

Summary A subline growing in medium without arginine and ornithine was established from a rat Reuber hepatoma cell line (R-Y121B·cho). The subline designated R-Y117B·cho was able to grow in glutamine, arginine and ornithine-free, glutamate-supplemented medium. Arginine synthesis from glutamate requires four urea cycle enzymes and another two enzymes, glutamate semialdehyde dehydrogenase and ornithine aminotransferase. Since R-Y121B·cho cells have all the urea cycle enzymes, two other enzyme activities were determined. The activities of ornithine aminotransferase and glutamate semialdehyde dehydrogenase were similar in R-Y117B·cho and its parental R-Y121B·cho cells, but R-Y117B·cho cells had higher conversion of glutamate to arginine than parental cells.     

Enzymes of amide and ureide biogenesis in developing soybean nodules

Amide and ureide biogenic enzymes were measured in the plant fraction of soybean (Glycine max) nodules during the period 11 to 23 days after inoculation with Rhizobium japonicum (USDA 3I1b142). Enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, and aspartate aminotransferase, showed substantial increases in their specific activities over the time course. These increases paralleled the induction of nitrogenase activity in the bacteroid and leghemoglobin synthesis in the plant fraction. The specific activity of asparagine synthetase, however, showed a rapid decline after an initial increase in specific activity. Following the initial increases in the ammonia assimilatory enzymes, there was an increase in the activity of 5-phosphoribosylpyrophosphate amidotransferase, the enzyme which catalyzes the first committed step of de novo purine biosynthesis. This was followed by a dramatic increase in the purine oxidative enzymes, xanthine dehydrogenase and uricase. Smaller increases were observed in the activities of enzymes associated with the supply of metabolites to the purine biosynthetic pathway: phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene tetrahydrofolate dehydrogenase.     

Cellular and Subcellular Localization of Hexokinase, Glutamate Dehydrogenase, and Alanine Aminotransferase in the Honeybee Drone Retina

Abstract: Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[³H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.     

Characterization of phospholipase A2 and acyltransferase activities in squid (loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Marker enzymes of Plasmodium falciparum and human erythrocytes as indicators of parasite purity

Plasmodium falciparum trophozoites, isolated by mechanical rupture of infected human erythrocytes, were analyzed for purity by determination of the specific activities of a number of marker enzymes selected for high activity, stability, and convenience of assay procedures. The specific activities of the soluble enzymes lactate dehydrogenase and malate dehydrogenase were much higher in the parasite than in the erythrocyte. The soluble enzyme glutamate dehydrogenase (NADP+) was specific for the parasite. Samples of 100,000 g supernate obtained from parasites that appeared to be free from contaminating erythrocytes consistently showed specific activities of about 4, 3 and 0.1 mumole/min/mg for lactate dehydrogenase, malate dehydrogenase and glutamate dehydrogenase, respectively. Moreover, preparations of parasites that exhibit these specific activities showed low acetylcholine esterase activity in the membrane fractions. The specific activities of these soluble marker enzymes did not appear to be strain dependent. A preparation of highly purified trophozoites obtained by free flow electrophoresis and analyzed for purity by electron microscopy exhibited the same specific activities for these marker enzymes. The use of specific activities of selected marker enzymes should be very useful for determining the purity of preparation of parasites when used in conjunction with other methods.