Glutamate producing aspartate aminotransferase in glutamatergic perforant path terminals of the rat hippocampus. Cytochemical and lesion studies

The enzyme aspartate aminotransferase was demonstrated cytochemically in the rat hippocampus 4, 7, and 14 days after unilateral entorhinal cortex lesion. At the light microscopic level the enzyme showed a significant activity decrease in the ipsilateral entorhinal terminal field which was similar at all postlesion times investigated. Non-denervated areas, i.e. the inner one-third of the dentate gyrus molecular layer and the radiatum layer of CA2/3, showed an increase of aminotransferase activities. At the electron microscopic level in the entorhinal terminal field of the control (unoperated) side aspartate aminotransferase was localized preferentially in a great number of boutons, containing the cytoplasmic and mitochondrial isoenzymes. Following entorhinal lesion a significant loss of these positively reacting boutons was seen. Most of the degenerating boutons contained reaction product but a small number was negative for aspartate aminotransferase. From 4 to 14 postlesion days the positively reacting boutons of the non-denervated supragranular zone expanded outward into the denervated area according to the known terminal proliferation of the commissural and associational systems. The remaining denervated entorhinal terminal field was reinnervated predominantly by negatively reacting boutons (probably terminal proliferations of septal afferents) and by a small number of positively reacting boutons (probably terminal proliferations of the crossed temporo-dentate pathway). The presence of cytoplasmic aspartate aminotransferase in the terminals of a well-known glutamatergic system is discussed in relation to the possible importance of this enzyme for the production of releasable glutamate.     

Cytochemical demonstration of aspartate aminotransferase in the mossy-fibre system of the rat hippocampus

A lead-salt procedure was used for the cytochemical demonstration of aspartate aminotransferase (AT) in the CA3 of the rat hippocampus. Cytoplasmic- and mitochondrial-AT isoenzymes were demonstrated in different neuronal structures, but not in astrocytes. Of special interest was the localization of cytoplasmic AT in most mossy-fibre boutons. This might indicate that cytoplasmic AT is responsible for the production of releasable glutamate in the glutamatergic/aspartatergic mossy fibre system.     

Cytochemical demonstration of aspartate aminotransferase in the mossy-fibre system of the rat hippocampus

Summary A lead-salt procedure was used for the cytochemical demonstration of aspartate aminotransferase (AT) in the CA3 of the rat hippocampus. Cytoplasmic- and mitochondrial-AT isoenzymes were demonstrated in different neuronal structures, but not in astrocytes. Of special interest was the localization of cytoplasmic AT in most mossy-fibre boutons. This might indicate that cytoplasmic AT is responsible for the production of releasable glutamate in the glutamatergic/aspartatergic mossy fibre system.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi.

1. The cultured, epimastigote-form of Trypanosoma cruzi contains NADP-linked glutamate dehydrogenase (EC 1.4.1.4), with a molecular weight of about 280,000, similar to the enzyme from Plasmodium chabaudi and different from the enzymes from higher animal sources. 2. T. cruzi also contains aspartate aminotransferase (EC 2.6.1.1), with properties similar to those of the enzyme from mammals. 3. The concerted action of the transaminase and glutamate dehydrogenase might be responsible for the production of NH3 which characterizes the protein catabolism in T. cruzi.     

Up regulation of calbindin-D28K mRNA in the rat hippocampus following focal stimulation of the perforant path

Calbindin-D28K is a constitutive Ca2(+)-binding protein expressed in hippocampal neurons that are resistant to various forms of excitotoxic injury. However, the local factors controlling calbindin-D28K expression within the central nervous system are unknown. We report that neuronal excitation via the perforant path leads to an increased expression of calbindin-D28K mRNA within dentate granule cells. This response is related specifically to stimulation that induces prolonged periods of bursting afterdischarges and precedes cellular injury. The up regulation of calbindin-D28K mRNA occurs during the type of neuronal activation associated with elevated cytosolic Ca2+ and suggests that the maintenance of Ca2+ homeostasis includes a system of feedback control at the level of gene expression.     

Degradation of aspartate aminotransferase in rat liver lysosomes

Highly purified lysosomes from the normal and leupeptin-treated rat livers were subjected to immunoblot analysis using antibodies against cytosolic and mitochondrial isozymes of aspartate aminotransferase (cAspAT and mAspAT). In the case of cAspAT (subunit M.W. = 46K), the leupeptin-treated lysosomes showed a major band of 46K and a minor band of 36K while normal lysosomes showed a major band of 36K and a minor band of 41K. In the case of mAspAT (subunit M.W. = 44K), the leupeptin-treated lysosomes showed a 44K band and the normal lysosomes showed a 40K band. These observations suggest that both cAspAT and mAspAT are sequestered into lysosomes with the original subunit molecular weights and are degraded in the lysosomes by way of sequential formation of relatively stable intermediates with distinct molecular weights.     

Glutamate and aspartate transport in rat brain mitochondria

1. Rat brain mitochondria did not swell in iso-osmotic solutions of ammonium or potassium (plus valinomycin) glutamate or aspartate, with or without addition of uncouplers. 2. Glutamate was able to reduce intramitochondrial NAD(P)(+); aspartate was able to cause partial re-oxidation. 3. These effects were inhibited by threo-hydroxy-aspartate in whole but not in lysed mitochondria. 4. The existence of a ;malate-aspartate shuttle' for the oxidation of extramitochondrial NADH was demonstrated. This shuttle requires the net exchange of glutamate for aspartate across the mitochondrial membrane. 5. Extramitochondrial glutamate did not inhibit intramitochondrial glutaminase under conditions in which the inhibition in lysed mitochondria was virtually complete. 6. The glutaminase activity of these mitochondria was not energy-dependent. 7. We conclude that these mitochondria do not possess a glutamate-hydroxyl antiporter similar to that of liver mitochondria nor a glutamate-glutamine antiporter similar to that of pig kidney mitochondria, but that they do possess a glutamate-aspartate antiporter.     

Denervation-induced spatiotemporal upregulation of ephrin-A2 in the mouse hippocampus after transections of the perforant path

Transections of the entorhinal afferent fibers to hippocampus, perforant path (PP), result in the denervation in specific hippocampal subregions, which is followed by a series of plastic events including axon sprouting and reactive synaptogenesis. Many growth-associated molecules are thought to participate in these events. In the present study, we proved the upregulation of ephrin-A2 in the denervated areas of the ipsilateral hippocampus following PP transections. Interestingly, when the elevation of ephrin-A2 reached the maximum axon sprouting in the denervated areas almost finished, implying the possible inhibitory effect of ephrin-A2 on sprouting. In addition, ephrin-A2 expression was observed in synapses during reactive synaptogenesis, suggesting that this molecule might also be implicated in the formation and maturation of synapses in the denervated areas.     

Acylation of aspartate aminotransferase

1. Acetylation of aspartate aminotransferase from pig heart inhibits completely the enzymic activity when the coenzyme is in the amino form (pyridoxamine phosphate) or when the coenzyme has been removed, but not when the coenzyme is in the aldehyde form (pyridoxal phosphate). 2. The group the acylation of which is responsible for the inhibition has been identified with the in-amino group of a lysine residue at the coenzyme-binding site. Moreover, in the pyridoxamine-enzyme the amino group of the coenzyme is also acetylated. 3. The reactivity of the coenzyme-binding lysine residue is greatly different in the pyridoxamine-enzyme and in the apoenzyme, suggesting the possibility of an interaction of its in-amino group with pyridoxamine or with other groups on the protein.     

Two forms of aspartate aminotransferase in rat liver and kidney mitochondria

1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.     

Effect of pyridoxine-deficiency on the turnover of aspartate aminotransferase isozymes in rat liver

DL-[14C]Leucine or L-[3H]leucine was injected intraperitoneally into pyridoxine-deficient and control rats, and the subsequent incorporation of the radioactivities into aspartate aminotransferase (AspAT) isozymes and the total soluble protein in the liver was measured. AspAT in the cytosol (AspATc) was separated into 3 subfractions with different characteristics on chromatofocusing. The results showed that in the liver of pyridoxine-deficient rats, the syntheses of all 3 subfractions of AspATc and degradation of AspATc (total) were increased, but that the syntheses and degradation of the total soluble protein and mitochondrial AspAT (AspATm) were not much different from those in control rats. The half-lives of soluble protein and AspATm were calculated to be 3.26-3.72 and 5.02-6.67 days, respectively, in both groups, and that of AspATc in control liver was found to be 4.78 days. The rate of degradation of AspATc in pyridoxine-deficient rat liver could not be calculated, because its kinetics were very complicated; there were apparently at least 2 components with different rates of degradation. Thus pyridoxal 5'-phosphate (PLP) apparently affects both the synthesis and degradation of AspATc, but does not affect the turnover of AspATm in rat liver.     

Conformation of aspartate aminotransferase in crystals

X-ray study of chicken cytosolic aspartate aminotransferase revealed conformational changes in the protein of two kinds: (1) a shift of the small domain adjacent to substrate-binding area due to interaction of the protein with two carboxyl groups of substrate and (2) a change in inclination of the coenzyme plane due to replacement of C = N bond of the coenzyme with Lys-258 by C = N bond with a substrate. An asymmetry in subunit behaviour is observed in both cases: the domain is shifted in one subunit and the coenzyme is rotated in other. Substrate-binding properties of each subunit are strictly dependent on the protein conformation in substrate-binding area.     

An electron microscope study on the termination of the perforant path fibres in the hippocampus and the fascia dentata

Summary Morphology and distribution of the perforant path fibres in the hippocampus and the fascia dentata of the rat have been studied in the electron microscope. Investigations were carried out on normal tissue as well as on tissue degenerating after entorhinal damage. The perforant path fibres were relatively thin and the terminals small. Two terminal fields were found to be of quantitative importance, one in the middle third of stratum moleculare of the fascia dentata, the other in stratum lacunosum moleculare of regio inferior of the hippocampus. Some of the observations have been expressed in numerical terms.This study was supported in part by Grant NB 02215 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.     

Distributions of aspartate aminotransferase and malate dehydrogenase activities in rat retinal layers

Aspartate aminotransferase (AAT), an enzyme interconverting glutamate and aspartate, has been suggested to be a marker for glutamatergic and/or aspartatergic neurons. However, AAT, glutamate, and aspartate are also involved in cellular metabolism, e.g., the malate-aspartate shuttle. To investigate the extent to which AAT might be involved in these several functions in retina, the distribution of AAT activity in rat retinal layers was compared to that of malate dehydrogenase (MDH), an enzyme of aerobic metabolism proposed to be physically complexed with AAT in the malate-aspartate shuttle mechanism. The distribution of AAT activity in retinal layers closely paralleled that of MDH (correlation coefficient AAT versus MDH = 0.93). AAT activity was proportionately higher than MDH in the photoreceptor inner segments, containing a high density of mitochondria, and in the outer plexiform layer (OPL), containing photoreceptor terminals and bipolar and horizontal cell processes. The amount of total AAT activity in the inner segments related to the mitochondrial isoenzyme is almost twice that in the other layers tested, including the OPL. The correlation between AAT and MDH activities is consistent with AAT involvement in retinal energy metabolism, although other functions, such as neurotransmission, are possible.     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.