Purification and characterization of a high-molecular-weight endogenous glutamate-binding inhibitor in porcine brain

A high-molecular-weight glutamate-binding inhibitor (HGBI) from porcine brain extract was purified to homogeneity. The results of this purification process show that glutamate receptor activity can be regulated by a high-molecular-weight protein, which inhibits [³H]L-glutamate binding to excitatory amino acid (EAA) receptors. The purified HGBI appears to be a protein with a molecular weight of approximately 70 kD. The purified HGBI is negatively charged, suggesting that it may contain acidic amino acids, and most likely,L-glutamate- andL-aspartate-enriched regions, responsible for its surface charge as well as for its binding to glutamate receptors. Inhibition of [³H]L-glutamate binding by the purified HGBI is reversible, and appears to change the binding kinetics. This endogenous ligand for glutamate receptors has unique characteristics separating it from all the other ligands found so far in the EAA receptor system. This HGBI represents a new class of modulator for the EAA receptor, thus further investigation of the function and structure of the HGBI should provide new understanding of the mechanisms of EAA-mediated neurotransmission.     

Tissue and regional distribution of cysteic acid decarboxylase

A sensitive and rapid assay method for cysteic acid decarboxylase was developed which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of³⁵S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.This work was supported in part by grant NS 13224 from the National Institutes of Health, U.S.A., and grant from Huntington's Chorea Foundation in memory of Mrs. Ruth Berman.This work was presented at the Eighth Annual Meeting of American Society for Neurochemistry in Denver, Colorado, March 1977.     

Taurine and central nervous system disorders

In the present era, investigators seek to find therapeutic interventions that are multifaceted in their mode of action. Such targets provide the most advantageous routes for addressing the multiplicity of pathophysiological avenues that lead to neuronal dysfunction and death observed in neurological disorders and neurodegenerative diseases. Taurine, an endogenous amino acid, exhibits a plethora of physiological functions in the central nervous system. In this review, we describe the mode of action of taurine and its clinical application in the neurological diseases: Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.     

Oxygen-induced seizures and inhibition of human glutamate decarboxylase and porcine cysteine sulfinic acid decarboxylase by oxygen and nitric oxide

The recombinant forms of the two human isozymes of glutamate decarboxylase, GAD65 and GAD67, are potently and reversibly inhibited by molecular oxygen (Ki = 0.46 and 0.29 mM, respectively). Inhibition of the vesicle-associated glutamate decarboxylase (GAD65) by molecular oxygen is likely to result in incomplete filling of synaptic vesicles with gamma-aminobutyric acid (GABA) and may be a contributing factor in the genesis of oxygen-induced seizures. Under anaerobic conditions, nitric oxide inhibits both GAD65 and GAD67 with comparable potency to molecular oxygen (Ki = 0.5 mM). Two forms of porcine cysteine sulfinic acid decarboxylase (CSADI and CSADII) are also sensitive to inhibition by molecular oxygen (Ki = 0.30 and 0.22 mM, respectively) and nitric oxide (Ki = 0.3 and 0.2 mM, respectively). Similar inhibition of glutamate decarboxylase and cysteine sulfinic acid decarboxylase by two different radical-containing compounds (O2 and NO) is consistent with the notion that these reactions proceed via radical mechanisms.     

Genistein and daidzein induce neurotoxicity at high concentrations in primary rat neuronal cultures

Summary It is known that estrogen can protect neurons from excitotoxicity. Since isoflavones possess estrogen-like activity, it is of interest to determine whether isoflavones can also protect neurons from glutamate-induced neuronal injury. Morphological observation and lactate dehydrogenase (LDH) release assay were used to estimate the cellular damage. It is surprising that, contrary to estrogen, isoflavones, specifically genistein and daidzein, are toxic to primary neuronal culture at high concentration. Treatment of neurons with 50 μM genistein and daidzein for 24 h increased LDH release by 90% and 67%, respectively, indicating a significant cellular damage. Under the same conditions, estrogen such as 17β-estradiol did not show any effect on primary culture of brain cells. At 100 μM, both genistein and daidzein increased LDH release by 2.6- and 3-fold, respectively with a 30-min incubation. Furthermore, both genistein and daidzein at 50 μM increased the intracellular calcium level, [Ca²⁺]_i, significantly. To determine their mode of action, genistein and daidzein were tested on glutamate and GABA_A receptor binding. Both genistein and daidzein were found to have little effect on glutamate receptor binding, while the binding of [³H]muscimol to GABA_A receptors was markedly inhibited. However, 17β-estradiol did not affect GABA_A receptor binding suggesting that the toxic effect of genistein and daidzein could be due to their inhibition of the GABA_A receptor resulting in further enhancement of excitation by glutamate and leading to cellular damage. Ying Jin, Heng Wu contributed equally to this article.     

Structural and functional analysis of cysteine residues in human glutamate decarboxylase 65 (GAD65) and GAD67

Previously, we have shown that brain glutamate decarboxylase (GAD) is greatly inhibited by sulfhydryl reactive reagent suggesting cysteine residue(s) may play an important role in GAD function. In this report, we determined the role of cysteine residues in the recombinant human 65-kDa GAD isoform (hGAD65) and 67-kDa GAD isoform (hGAD67), using a combination of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and site-directed mutagenesis. Here, we report that cysteine 446 (C446) in hGAD65 is important for its activity and is present as free sulfhydryl group. This conclusion is based on the following observations: (i) mutation of C446 in hGAD65 to alanine reduced hGAD65 activity by more than 90%, (ii) MALDI-TOF analysis of the non-reduced, trypsin-digested GAD65 revealed that C446 is present as a free sulfhydryl group as indicated by a peak at m/z (mass/charge) 647.3446 (peptide 443-448) and, when GAD65 was treated with sulfhydryl reagent, N-ethylmaleimide (NEM), the peak is shifted to m/z 772.3702,a mass increase of 125.1 daltons (Da) as a result of modification of cysteine by NEM. Parallel studies have also been conducted with hGAD67. Cysteine 455 was found to be important for GAD67 activity.     

Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.     

Distribution and tissue specificity of 4-aminobutyrate-2-oxoglutarate aminotransferase

A rapid and specific method for assaying 4-aminobutyrate-2-oxoglutarate aminotransferase was developed. The method was based on the selectivity of ion exchange resin and the speed of vacuum filtration. With this new method, the aminotransferase activity in various tissues has been determined as follows: brain, 10.2; spinal cord, 11.8; liver, 5.7; kidney, 4.6; heart, 0.5; lung, 0.4 nmol glutamate formed/min/mg. No activity could be detected in muscle preparations. When the aminotransferases were tested with the antibody, against the purified 4-aminobutyrate aminotransferase from brain, no difference could be detected among brain, spinal cord, and kidney preparations as judged from the results of immunodiffusion, inhibition of enzyme activity by antibody, and microcomplement fixation. It is concluded that 4-aminobutyrate aminotransferases from various tissues of the mouse are probably identical or closely related.This work was supported in part by Grant No. NS 13224 and P01 NS 12116 from the National Institutes of Health, U.S.A. and Grant from Huntington's Chorea Foundation.All correspondence should be addressed to Dr. Wu.     

Abnormal protein patterns in multiple sclerosis

Sera from patients with multiple sclerosis (MS) and those obtained from normal subjects are indistinguishable by regular 5% or 7% polyacrylamide gel electrophoresis. However, 11 out of 15 MS sera examined by gradient polyacrylamide gel electrophoresis showed three distinct protein bands. None of the sera obtained from 10 normal subjects showed the characteristic protein patterns when they were examined by gradient gel electrophoresis. Similar results were obtained with de-albumin serum samples or with serum proteins precipitable at 50% ammonium sulfate saturation. These three proteins have now been purified to homogeneity by preparative gradient gel electrophoresis. Molecular weights of these proteins were estimated from gradient gel electrophoresis as 398,000, 363,000, and 302,000 daltons, respectively.This work was presented at the Tenth Annual Meeting of American Society for Neurochemistry on March 12, 1979, in Charleston, South Carolina.     

Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABA_A receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABA_A receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [³H]FNZP binding.