Biochimica et Biophysica Acta (BBA) - General Subjects
Theanine, γ-glutamylethylamide, is metabolized by renal phosphate-independent glutaminase
Introduction
Theanine, γ-glutamylethylamide, is an amino acid derivative found in Japanese green tea leaves (Camellia sinensis). It was first isolated and identified in the late 1940s by Sakato [1]. The other natural source of theanine so far discovered is the mushroom Xerocomus badius [2]. Although most amino acids are found at lower levels, theanine, which renders the gracefully sweet taste, accounts for more than 50% of total free amino acids in tea. The content of theanine in tea is between 1.5% and 3% (on average) of the dry weight [3], [4].
Regarding the physiological functions of theanine, many researchers have reported significant reduction in blood pressure in spontaneously hypertensive rats after some amounts of tea extract were ingested [5], relief from convulsion [6], [7], [8] or excitation [9] induced by caffeine ingestion, an influence on the brain levels of norepinephrine, serotonin, 5-hydroxyindole acetic acid and dopamine [10], [11], [12], and a protecting effect of brain tissue from glutamate neurotoxicity [13], [14].
A previous study demonstrated that significant amounts of ethylamine were detected in human urine collected after ingestion of an appreciable amount of tea extract, presumably as the result of the hydrolysis of theanine [15]. We have preliminarily reported that the kidney would seem to be the most expected site responsible for the enzymatic hydrolysis of theanine [16]. Now we have tried to purify and characterize the enzyme responsible for the degradation of theanine.
Section snippets
Chemicals
l-Theanine and l-γ-glutamyl-p-nitroanilide used in this experiment were commercial products from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). l-Glutamyl-γ-methylamide, l-glutamyl-γ-ethylester and l-glutamyl-γ-methylester were obtained from ICN Pharmaceuticals Japan (Tokyo, Japan). NAD-dependent glutamate dehydrogenase preparation [EC 1.4.1.2] (from beef liver) was obtained from Oriental Yeast Co., Ltd. (Tokyo, Japan). Reduced glutathione (GSH), bromelain (from pineapple stem) and l
Distribution of theanine-degrading enzyme in rat tissues
Each homogenate (20 μl) prepared from brain, thymus, heart, lung, liver, kidney, muscle, spleen, small intestine and blood plasma, respectively, was used as the enzyme source. When the theanine-degrading activity was tested in terms of glutamate formation, this activity was exclusively detected in kidney homogenate. No theanine-degrading activity was found from other tissue homogenate. Glutaminase activity, which was measured using the reaction medium described for the phosphate-independent
Discussion
Mammalian glutaminase [EC 3.5.1.2] was extensively studied in the 1970s [17], [18], [19], [20], [21], [22], [23], [24]. At that time, it was elucidated that there were at least two isozymes in kidney, brain and other tissues; one is phosphate-dependent glutaminase [20], [21] and the other is phosphate-independent glutaminase [17], [18], which possesses γ-glutamyl transpeptidase activity [21], [22], [23], [24]. However, it has never been examined whether or not theanine can be used as substrate
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