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M B Robinson

Publications and source records attributed to M B Robinson.

At least 19 recordsLinked to original sources
PubMed1994

An animal model of early-treated PKU.

Phenylketonuria (PKU) is a genetic disorder in which the hydroxylation of phenylalanine (Phe) to tyrosine is severely disrupted. If PKU is left untreated, severe mental retardation results. The accepted treatment is to restrict dietary intake of Phe. It has generally been thought that cognitive impairments are prevented if levels of Phe in plasma are maintained at or below five times the normal level. However, we recently documented that children treated early and continuously for PKU or children mildly hyperphenylalaninemic, who have levels of Phe in plasma approximately three to five times normal, still have cognitive impairments. These impairments are specific to the functions of frontal cortex (A. Diamond, W. Hurwitz, E. Lee, W. Grover, and C. Minarcik, unpublished observations). To investigate the mechanism underlying these cognitive deficits, an animal model of this condition was developed and characterized. Thirty-six rat pups were divided into three groups. The first group was treated pre- and postnatally with Phe and alpha-methylphenylalanine (a phenylalanine hydroxylase inhibitor). The second group was injected postnatally with Phe and alpha-methylphenylalanine. The third group received postnatal control injections. The mild plasma Phe elevations in the two experimental groups produced significant behavioral and neurochemical effects. Both experimental groups were impaired on a task dependent on frontal cortex, delayed alternation. Levels of dopamine, homovanillic acid (HVA), norepinephrine, and 5-hydroxyindole acetic acid (5-HIAA) were measured in medial prefrontal cortex, anterior cingulate cortex, striatum, and nucleus accumbens. The largest neurochemical reductions observed were in HVA and were in the two frontal cortical areas (medial prefrontal cortex and anterior cingulate cortex). There were modest reductions in HVA in the nucleus accumbens but no significant changes in HVA, or in any other metabolite or neurotransmitter, in the striatum. The levels of 5-HIAA were also reduced in all brain regions examined. There was no effect on norepinephrine in any of the four regions examined. Reduced levels of HVA in medial prefrontal cortex were the only neurochemical effect that significantly correlated with every measure of performance on the delayed alternation task. This study provides evidence of deleterious effects from mild elevations in the levels of Phe in plasma previously considered small enough to be safe. These effects include impaired performance on a cognitive task dependent on frontal cortex and reduced HVA levels in frontal cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Inhibition of glutamate uptake with L-trans-pyrrolidine-2,4-dicarboxylate potentiates glutamate toxicity in primary hippocampal cultures.

Sodium-dependent, high-affinity glutamate transport is generally assumed to limit the toxicity of glutamate in vivo and in vitro, but there is very little direct evidence to support this hypothesis. In the present study, the effects of the specific uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate on the toxicity and clearance of glutamate were examined in hippocampal neuronal cultures. At a concentration that was not toxic by itself, L-trans-pyrrolidine-2,4-dicarboxylate increased the toxicity of glutamate approximately fivefold and slowed the clearance of glutamate from the extracellular space. This toxicity was almost completely blocked by the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These studies provide direct evidence that sodium-dependent, high-affinity glutamate transport limits glutamate toxicity in vitro.

Animals↗
PubMed1992

Brain serotonin2 and serotonin1A receptors are altered in the congenitally hyperammonemic sparse fur mouse.

In previous studies we documented an increase in the levels of the serotonin metabolite, 5-hydroxyindoleacetic acid, in the congenitally hyperammonemic sparse fur mouse. To extend these findings, brain serotonin receptors were studied in these animals. Radioligand binding assays were performed using [3H]ketanserin to label serotonin2 sites and 8-[3H]hydroxy(di-n-propylamino)tetralin to label serotonin1A sites in cortical membrane homogenates. The capacity (Bmax) for [3H]ketanserin binding was significantly lower (-21%; p less than 0.05) in sparse fur animals than in control animals; there was no change in affinity (KD). In contrast, the capacity for 8-[3H]hydroxy(di-n-propylamino)tetralin binding was significantly greater (26%; p less than 0.05) in sparse fur compared with control animals. No difference in affinity was observed. Using two behavioral assays, the functional responsiveness of these serotonin receptors was compared in sparse fur and control animals. Head twitch activity elicited by administration of the serotonin agonist quipazine was studied as a behavior mediated by serotonin2 receptors. Compared with controls, sparse fur mice demonstrated a significantly decreased head twitch response (p less than 0.005). Hypothermia elicited by administration of 8-hydroxy(di-n-propylamino)tetralin was studied as a physiologic response mediated by serotonin1A receptors. Although there were not overall group differences in the dose-response data, there was a significant increase in the hypothermia induced by 8-hydroxy(di-n-propylamino)tetralin in sparse fur compared with control mice (p less than 0.02) at the highest dose. These data provide further support for a link between hyperammonemia and alterations in the serotonin system.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Multiple mechanisms for inhibition of excitatory amino acid receptors coupled to phosphoinositide hydrolysis.

Excitatory amino acid (EAA) analogues activate receptors that are coupled to the increased hydrolysis of phosphoinositides (PIs). In these studies, hippocampal slices were prepared from neonatal rats (6-11 days old) to characterize the effects of EAA analogues on these receptors. The concentrations of ibotenate and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate (trans-ACPD) required to evoke half-maximal stimulation (EC50 values) were 28 and 51 microM, respectively. Although the data for stimulation of PI hydrolysis by ibotenate and trans-ACPD were best fit to theoretical curves that had Hill slopes of 1, data for stimulation of PI hydrolysis by quisqualate were best fit to two sites. The EC50 values were 0.43 and 44 microM. The high-affinity sites were 70% of the total. A number of EAA analogues were tested for inhibition of PI metabolism. One of these, L-aspartate-beta-hydroxamate (L-A beta HA), was identified as a novel inhibitor of this response. L-A beta HA was equipotent as an inhibitor of PI metabolism stimulated by ibotenate, quisqualate, and trans-ACPD. The data for this inhibition were best fit to two sites. Between 32 and 48% of the total sites had high affinity with IC50 values in the range of 1.2-6.3 microM. The low-affinity sites had IC50 values between 610 and 2,700 microM. DL-2-Amino-3-phosphonopropionate (DL-AP3) was also equipotent as an inhibitor of PI hydrolysis stimulated by ibotenate, quisqualate, and trans-ACPD (IC50 values were 480-850 microM). In contrast to the data for L-A beta HA, the data for DL-AP3 were best fit to a single site. Both of these inhibitors reduced the maximal response caused by the agonists, consistent with noncompetitive mechanisms of action. Several experiments were designed to examine potential mechanisms for these noncompetitive effects. These studies suggest that either L-A beta HA and DL-AP3 bind to a site on the receptor and irreversibly block activation of the receptor, or that these inhibitors act via a distinct site that specifically regulates EAA receptors coupled to PI hydrolysis.

Alanine↗
PubMed1992

Quinolinate in brain and cerebrospinal fluid in rat models of congenital hyperammonemia.

Children with inborn errors of urea synthesis who survive neonatal hyperammonemic coma commonly exhibit cognitive deficits and neurologic abnormalities. Yet, there is evidence that ammonia is not the only neurotoxin. Hyperammonemia appears to induce a number of neurochemical alterations. In rodent models of hyperammonemia, uptake of L-tryptophan into brain is increased. It has been reported that in an experimental rat model of hepatic encephalopathy, in the ammonium acetate-injected rat, and in patients with hepatic failure and inborn errors of ammonia metabolism, quinolinate, a tryptophan metabolite, is increased. Elevations in quinolinate are of particular concern, as quinolinate could excessively activate the N-methyl-D-aspartate subclass of excitatory amino acid receptors, thereby causing selective neuronal necrosis. We sought to identify an animal model that would replicate the increases in quinolinate that have been associated with hyperammonemia in humans. Levels of quinolinate were measured in hyperammonemic urease-infused rats and ammonium acetate-injected rats. In the urease-infused rat, brain tryptophan was doubled, and serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly increased. Yet, despite the increase in tryptophan and evidence for increased metabolism of tryptophan to serotonin, there were no observed increases of quinolinate in brain, cerebrospinal fluid, or plasma. In the ammonium acetate-injected rat, significant increases of 5-hydroxyindoleacetic acid in cerebral cortex were also observed, but quinolinate did not change in cerebrospinal fluid or cerebral cortex. In summary, we were unable to demonstrate an increase of quinolinate in brain or cerebrospinal fluid in these rat models of hyperammonemia.

Acetates↗
PubMed1991

Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain.

The transport of L-[3H]glutamate into crude synaptosomal membrane fractions prepared from cerebellum, brainstem, hippocampus, cortex, striatum, and midbrain was characterized. In all brain regions, greater than 95% of the accumulation of radiolabel was sodium-dependent and the concentration-dependence was consistent with a single high affinity site. Dihydrokainate and L-alpha-aminoadipate were region specific inhibitors of uptake; this inhibition was consistent with a competitive mechanism. In the forebrain regions examined, dihydrokainate inhibited transport with IC50s of approx. 100 microM (range from 80 to 170 microM). Transport in cerebellum was essentially dihydrokainate-insensitive L-alpha-Aminoadipate inhibited transport in forebrain regions with IC50s of approx. 700 microM (range from 590 to 800 microM) and inhibited transport in cerebellum with an IC50 of 40 microM. The inhibition data obtained with forebrain and cerebellar tissues were consistent with nearly homogeneous (greater than 80%) populations of non-interacting sites. Inhibition data obtained with tissue prepared from brainstem were best fit to a mixture of the two sites (35-50% of the type observed in cerebellum). Other previously identified uptake inhibitors, including DL-threo-hydroxyaspartate, L-aspartate-beta-hydroxamate, beta-glutamate, and L-cysteine sulfinate were not selective for the two types of transport. These data demonstrate that there are two pharmacologically distinct sodium-dependent high affinity transport systems with heterogeneous regional distributions.

Amino Acids↗
PubMed1990

Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.

Animals↗
PubMed1989

Seizures decrease regional enzymatic hydrolysis of N-acetyl-aspartylglutamate in rat brain.

Previous results have shown that kindled seizures increase N-acetyl-aspartylglutamate (NAAG) levels in the entorhinal cortex, while non-kindled convulsions have no effect. To further explore possible relationships between epilepsy and the physiology of NAAG, the effect of amygdaloid kindling on the activity of a NAAG-hydrolyzing enzyme was examined in specific brain regions associated with limbic seizures. NAAG is hydrolyzed into glutamate (Glu) and N-acetyl-aspartate (NAA) by N-acetylated-alpha-linked acidic dipeptidase (NAALADase), a membrane-bound peptidase. We found that convulsions decreased NAALADase activity and these effects were generalized to several brain regions. While small decreases in the hippocampus were specific to kindling, the decreases in other limbic regions were larger, non-specific, and appear to be aftereffects of convulsions; i.e. not specific to kindling. Although there is evidence that NAAG may be an excitatory neurotransmitter, it could also function as a storage form of Glu. Thus, a reduction in NAALADase activity could reduce the availability of Glu at certain synapses, which might be a homeostatic mechanism for lessening susceptibility to further seizures.

Animals↗
PubMed1989

The effects of N-acetylated alpha-linked acidic dipeptidase (NAALADase) inhibitors on [3H]NAAG catabolism in vivo.

N-Acetylated, alpha-linked acidic dipeptidase (NAALADase) is a chloride-activated, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N-acetyl-aspartyl-glutamate (NAAG) in vitro. To determine whether NAALADase is the catabolic enzyme of NAAG in vivo, we have examined the effects on [3H]NAAG metabolism of intrastriatal co-injections of agents that affect NAALADase activity in vitro. Co-injections of NAALADase inhibitors, such as quisqualate (Quis), phosphate, dithiothreitol and EGTA were found to prolong the t1/2 of [3H]NAAG, whereas cobalt, a NAALADase activity stimulator, accelerated [3H]NAAG catabolism. These results are consistent with a role for NAALADase in the extracellular disposition of endogenous NAAG.

Animals↗
PubMed1988

A re-examination of the interaction of N-acetyl-L-aspartyl-L-glutamate with a subpopulation of rat brain membrane L-[3H]glutamate binding sites.

The neuropeptide, N-acetyl-L-aspartyl-L-glutamate (NAAG) has been reported to act at a subpopulation of putative quisqualate receptors on the basis of its competitive inhibition of specific binding of L-[3H]glutamate and on the basis of quisqualate-sensitive binding of [3H]NAAG radiolabeled on the glutamate moiety. Recently, a membrane-bound metallopeptidase, N-acetylated alpha-linked acidic dipeptidase (NAALADase), which cleaves NAAG to N-acetyl-aspartate (NAA) and glutamate, has been characterized and has been shown to exhibit optimal activity under incubation conditions used to measure NAAG binding sites. Accordingly, we have examined whether NAALADase mediated release of glutamate from NAAG might account for the receptor binding results. Insertion of empirically derived kinetic constants for NAALADase hydrolysis of NAAG into a theoretical model for peptide-derived glutamate inhibition of [3H]glutamate binding reveals that NAAG can appear to displace, with high affinity, a subpopulation of [3H]glutamate labeled sites, and yet have little or no intrinsic activity for these sites. Furthermore, empirical data relating time and protein concentration to NAAG displacement of [3H]glutamate binding are more consistent with a proteolytic mechanism rather than an equilibrium interaction of the peptide with membrane receptors. Coupled with recent findings attributing the Cl- -dependent glutamate binding to a sequestration phenomenon, these results demonstrate that the inferred action of NAAG at glutamate synaptic receptors through previous radioligand binding studies is probably incorrect. Furthermore, these studies offer a general caution regarding the conclusions about subpopulations of receptors drawn from receptor binding assays conducted with ligands, which may be structurally modified by enzymatic processes.

Animals↗
PubMed1988

Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues: findings with a novel liquid phase radioimmunoassay.

Antibodies were raised in rabbits against the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes.

To permit a molecular characterization of neurotransmitter transporter proteins, we have studied uptake activities induced in Xenopus laevis oocytes after injection of adult rat forebrain, cerebellum, brainstem, and spinal cord poly(A)+ RNA. L-Glutamate uptake could be observed as early as 24 hr after injection, was linearly related to the quantity of mRNA injected, and could be induced after injection of as little as 1 ng of cerebellar mRNA. Transport of radiolabeled L-glutamate, gamma-aminobutyric acid, glycine, dopamine, serotonin, and choline could be measured in single microinjected oocytes with a regional profile consistent with the anatomical distribution of particular neurotransmitter synthesizing soma. Forebrain L-glutamate and dopamine uptake, as well as cerebellar L-glutamate transport, were found to be Na+-dependent. Cerebellar mRNA-induced L-glutamate transport was both time and temperature-dependent, was saturable by substrate, suggesting a single activity with an apparent transport Km of 14.2 microM and a Vmax of 15.2 pmol/hr per oocyte, and was sensitive to inhibitors of brain L-glutamate transport. Thus, the oocyte L-glutamate transport induced by injection of adult rat cerebellar mRNA appears essentially identical to the high-affinity, Na+-dependent L-glutamate uptake found in brain slices and nerve terminals. Experiments with size-fractionated cerebellar mRNA reveal single, comigrating peaks for cerebellar L-glutamate and gamma-aminobutyric acid transport, with peak activity obtained in fractions of approximately 2.7 kilobases, suggesting the presence of single or similarly sized mRNAs encoding each of these activities.

Animals↗
PubMed1988

Calcium-dependent evoked release of N-[3H]acetylaspartylglutamate from the optic pathway.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide localized to several putative glutamatergic neuronal systems, including the rodent optic pathway. To determine whether the peptide is released by depolarization, the superior colliculus of the rat was perfused with 2 microCi of [3H]NAAG, then with Krebs-bicarbonate buffer for 1 h, using a microdialysis system. Subsequently, 10-min fractions were collected and analyzed by HPLC for [3H]NAAG. Addition of 100 microM veratridine resulted in a several-fold increase in the evoked release of [3H]NAAG that was virtually abolished by coperfusion with Ca2+-free Krebs buffer containing 1 mM EGTA. When [3H]glutamate was used as the precursor, veratridine depolarization resulted in only an 80% increase in the release of [3H]NAAG. Prior enucleation of the right eye reduced the spontaneous release of [3H]NAAG by 50%, and the veratridine-evoked release by greater than 85%, from the left superior colliculus. These results suggest that NAAG is released upon depolarization and may serve as a neurotransmitter/neuromodulator in the optic tract.

Animals↗
PubMed1988

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate: subcellular and regional distribution, ontogeny, and the effect of lesions on N-acetylated-alpha-linked acidic dipeptidase activity.

N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a Cl- dependent, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) in vitro. To examine the pattern of NAALADase expression in the CNS, subcellular, regional, and developmental studies were conducted. Subcellular fractionation of lysed synaptosomal membranes revealed a substantial enrichment of the peptidase in synaptic plasma membranes as compared to mitochondrial or myelin subfractions. Regional studies reveal an apparent restriction of peptidase activity to kidney and brain. A threefold variation in specific activity was observed among brain regions, with highest specific activity in the cerebellum and lowest in telencephalic structures, a pattern that does not, in general, correlate with NAAG levels. Ontogenetic studies demonstrate a region-dependent, postnatal pattern of expression of NAALADase activity, with adult levels attained earliest in brainstem, as was previously reported for NAAG. Postnatal NAALADase expression would not appear to support a role for the peptidase in constitutive protein processing, but rather suggests that NAALADase may play a role in synaptic peptide degradation. Glutamate (Glu) excised from NAAG by NAALADase could be transported efficiently by uptake processes. Lesion studies, however, do not support a close structural association between NAALADase activity and the corticostriatal sodium-dependent, high-affinity, Glu uptake system. Similar to in vitro data documenting the route of NAAG degradation by NAALADase, after intrastriatal injection, NAAG was rapidly cleaved to two major products, N-acetyl-aspartate and Glu, with a t1/2 of approximately 10 min. Thus, the route of in vivo catabolism of NAAG parallels results from studies on NAALADase activity in vitro. These results are consistent with a role of NAALADase in the synaptic processing of NAAG. However, certain discrepancies in the regional and ontogenetic profiles of NAAG and NAALADase suggest that this relationship is not an exclusive one and may reflect a role for NAALADase on additional N-acetylated acidic peptides in vivo.

Animals↗
PubMed1988

Effect of sodium benzoate and sodium phenylacetate on brain serotonin turnover in the ornithine transcarbamylase-deficient sparse-fur mouse.

Herein we examine the effects of sodium benzoate and sodium phenylacetate on feeding and central serotonin turnover in a child with citrullinemia and in an animal model of congenital hyperammonemia, the ornithine transcarbamylase-deficient sparse-fur (spf/y) mouse. In the child, when the benzoate/phenylacetate dosage was increased from 200 to 375 mg/kg/day each, feeding decreased. There was an accumulation of benzoate and phenylacetate in blood and cerebrospinal fluid as well as an increased concentration of 5-hydroxyindoleacetic acid, a neurochemical marker for serotonin turnover, in cerebrospinal fluid. In the mouse, sodium benzoate had a biphasic effect on both plasma ammonium levels and brain serotonin turnover. Two percent oral benzoate was associated with an increase in ammonium level, while a 3% dose led to a decrease in ammonium. There was a similar effect on serotonin turnover noted in both the hyperammonemic spf/y and control CD-1/y mice. Sodium phenylacetate did not have a consistent effect on serotonin turnover. The mechanism by which benzoate increases brain serotonin turnover appears to involve competition with tryptophan for albumin binding sites. This results in increased free tryptophan in serum and brain. We speculate that some of the clinical symptoms of benzoate intoxication may be a consequence of altered serotonin turnover in the brain. We suggest that drug levels be monitored during therapy.

Administration, Oral↗
PubMed1987

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain.

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.

Animals↗