A fl uorescence enhancement assay for measurement of glutamate decarboxylase activity

Glutamate (Glu) is the main excitatory and -aminobutyric acid (GABA) the main inhibitory neurotransmitter in the mammalian nervous system. Excessive levels of extracellular Glu in the nervous system are excitotoxic and lead to several neurodegenerative processes [1-3]. Conversely, GABA is known to have several physiopathological functions such as epilepsy, anti-anxiety, and anti-diabetic effect in humans [4-7]. The Glu and GABA have a complex homeostatic relationship that brings balance to the level of brain activity. In GABAergic neurons, Glu is converted into GABA by glutamate decarboxylase (GAD, EC 4.1.1.15), the rate-limiting enzyme in the synthesis of GABA. The enzyme is present in several non-neuronal tissues including the pancreatic -cells, [8-11]. The enzyme exists as two isoforms, named GAD67 and GAD65 [12,13]. GAD67 is essentially active, while GAD65 can be activated in response to an additional demand for extra GABA in neurotransmission [14,15]. GAD65 exists mainly in an inactive form that can be activated by its coenzyme, pyridoxal 5’-phosphate (PLP) [16]. It has been demonstrated that there are many similarities in identity of rat brain GAD 65 with the human GAD 67 [17]. Since the discovery of GAD as an antigen in Type 1 Diabetes (T1D) several analytical methods have been described for the detection of its anti-GAD antibodies [18]. However, regulation of GAD activity in pancreatic -cells plays a key role in governing the cell function for production and secretion of insulin [10,19]. There are still many unanswered questions that need to be investigated in the regulation of GAD activities in the therapy of both T1D and type 2 diabetes (T2D). Considerable studies support the evidence regarding the localization of GAD and physiological function of GABA, given the original suggestion that certain human pathological disorders is associated with the alteration in GAD activity both in central and peripheral disorders. Despite its importance, the precise mechanism underlying the regulation of GAD activity and the chemical mechanisms for PLP-mediated reactions with an emphasis on the chemical steps processed by enzyme in the specifi c cells Abstract


Introduction
Glutamate (Glu) is the main excitatory and -aminobutyric acid (GABA) the main inhibitory neurotransmitter in the mammalian nervous system. Excessive levels of extracellular Glu in the nervous system are excitotoxic and lead to several neurodegenerative processes [1][2][3]. Conversely, GABA is known to have several physiopathological functions such as epilepsy, anti-anxiety, and anti-diabetic effect in humans [4][5][6][7]. The Glu and GABA have a complex homeostatic relationship that brings balance to the level of brain activity. In GABAergic neurons, Glu is converted into GABA by glutamate decarboxylase (GAD, EC 4.1.1.15), the rate-limiting enzyme in the synthesis of GABA. The enzyme is present in several non-neuronal tissues including the pancreatic -cells, [8][9][10][11]. The enzyme exists as two isoforms, named GAD67 and GAD65 [12,13]. GAD67 is essentially active, while GAD65 can be activated in response to an additional demand for extra GABA in neurotransmission [14,15]. GAD65 exists mainly in an inactive form that can be activated by its coenzyme, pyridoxal 5'-phosphate (PLP) [16].
It has been demonstrated that there are many similarities in identity of rat brain GAD 65 with the human GAD 67 [17]. Since the discovery of GAD as an antigen in Type 1 Diabetes (T1D) several analytical methods have been described for the detection of its anti-GAD antibodies [18]. However, regulation of GAD activity in pancreatic -cells plays a key role in governing the cell function for production and secretion of insulin [10,19].
There are still many unanswered questions that need to be investigated in the regulation of GAD activities in the therapy of both T1D and type 2 diabetes (T2D). Considerable studies support the evidence regarding the localization of GAD and physiological function of GABA, given the original suggestion that certain human pathological disorders is associated with the alteration in GAD activity both in central and peripheral disorders. Despite its importance, the precise mechanism underlying the regulation of GAD activity and the chemical Citation: Messripour [20] sequential enzymatic reactions for conversion of GABA to succinate [21] or HPLC measurement of GABA production [22,23]. These methods need complex chemical substances or radiolabeled materials that are often expensive and/or health hazardous.
For clinical diagnostic purposes and pharmaceutical industries a simpler, more sensitive and reliable enzyme assay is needed to clearly delineate the enzymatic activity and metabolic roles of the enzyme in tissue samples.
The interaction between the phosphate group of PLP and the active site of GAD, maintains PLP molecule in the fl uorogenic site that upon addition of glutamate conformational changes can be detected fl uorimetrically [24]. Because of the rate of fl uorescence emission of GAD is likely limited upon addition its substrate the present study extends the initial works on the use of the fl uorimetric properties to determine GAD activity in the small amounts of biological samples.

Purifi cation of glutamate decarboxylase
Purifi cation of rat brain GAD was essentially carried out as described by Nathan, et.al. [25], briefl y, in each experiment,

Manometric measurement of GAD activity
In order to compare the results of the fl uorimetric method with a non-fl uorimetric one, conventional mercury manometric technique (Warburg) was chosen. The most commonly measured end product has been CO2, and this gas has been measured by techniques which include Warburg manometry [27]. Briefl y, 1 ml aliquot of the brain supernatant or eluted partially purifi ed enzyme preparations with protein concentration as described above were adjusted to pH 7 and transferred in the manometric cell and incubated at 37°C for 5 min. The reaction was then started by the addition of 100μl glutamate solution (10 mM) and CO2 production was measured for 15 min. The results are expressed as μlCO2 released/min/ mg protein.

Fluorimetric measurement of GAD activity
In the fl uorimetric method, 1 ml aliquot of the brain supernatant or eluted partially purifi ed enzyme preparations with protein concentrations as shown in Table 1

Results
The specifi c activity of GAD in rat brain supernatant and the fractions eluted from each chromatographic step as measured by two different methods a Warburg manometric method and a fl uorimetric method. Data are summarized in Table 1. Both fl uorescence emission and CO2 release of the experimental mixture containing tissue preparations were markedly increased after the addition of glutamate. Whereas, the total protein content of the supernatant that applied to the three chromatographic purifi cation steps was 640 mg which recovered from the last chromatographic step was 0.67 mg.
which its specifi c enzyme activity increased approximately 140 folds of that in the supernatant. This is in good agreement with the results previously reported [25]. As shown in Table 1  containing tissue preparations were markedly increased after the addition of glutamate. During effi cient purifi cation steps, specifi c activity as expressed by fl uorescence emission or CO2 release/mg of protein signifi cantly increased, indicating that the GAD protein is getting more abundant ( Table 1). The increase in the purifi cation folds of the enzyme as measured by manometric and fl uorimetric techniques were quite similar.
The rate of CO2 production and the changes of fl uorescence intensities in each step of purifi cation were measured by manometric and fl uorimetric methods. The results are mean of 6 separate experiments with SD in round brackets. In each experiment 5 forebrains were processed as described in the Method section.

Discussion
The results reported in this paper demonstrated that the release of CO2 and the changes of the fl uorescence intensities (ΔF) of the assay mixture increased markedly as purifi cation proceeded ( Table 1). The increasing of the fl uorescence intensities of the purifi ed GAD upon the addition of glutamate indicates the formation of GAD/glutamate complex [24]. Changes in fl uorescence intensities (ΔF)/min/mg protein by the supernatant and the eluted fractions from three chromatographic steps of purifi cations positively correlated with the levels of μlCO2 released/min/mg protein CO2 of the enzyme preparations(r=99%). However, the results in Table  1 showed that the levels of Standard Deviation (SD) related to the specifi c CO2 production of manometric method to its corresponding values of specifi c ΔF obtained from the fl uorimetric technique was markedly higher. This suggests a higher sensitivity for the fl uorimetric method compared to the manometric method. There are several methods for the determination of GAD activity using different procedures and some of these studies are reporting the advantageous properties of fl uorimetric methods over the other ones [28][29][30][31]. The results of this study providing further support for the previous report that the fl uorimetric method has advantages of being easy and rapid, with higher quantitative sensitivity and reliability as compared with the conventional manometric method which involved CO2 determination.. In conclusion, the fl uorometric assay of GAD seems to be the most accessible, simple with low-cost and may be used in clinical and pharmaceutical investigations.