1.1 1., Litopenaeus vannamei N- -β-d- NAGase. Asp Glu. K I 9.50 mmol/l mmol/l. Litopenaeus vannamei

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1 N--β-D- 1, , GlyAlaValLeu Ile Pro PheTrpSerThr Cys Gln AsnGlu AspHis LysArg 18 Litopenaeus vannamei N--β-D- pnp-nag NAGase L-His Lys Arg Asp Glu IC mmol/l Asp Glu pnp-nag Asp Glu K I 9.50 mmol/l mmol/l Litopenaeus vannamein--β-d- Q A (2006) N- -β-d- NAGase EC [1] NAGase [2] NAGase [3~5] [6~8] NAGase Litopenaeus vannamei NAGase NAGase 1.1 N--β-D- [9] PAGE 1560 U/mg -N--β-D- pnp-β-d-glcnac (2004J054); (JA04260) (1974 ) ; E mail: chenqx@jingxian.xmu.edu.cn 46 /2006 / 30 / 3

2 1.2 [10] pnp-β-d-glcnac 2 ml 0.15 mol/l NaAc-HAc (ph 5.2) 0.5 mmol/l min 2 ml 0.5 mol/l NaOH 752 Spectrum 405 nm (A 405nm ) L/(mol cm) [11] Lineweaver-Burk 2.1 NAGase GlyAlaVal Leu IlePro PheTrpSer Thr Cys Gln AsnGlu Asp HisLysArg ~15 mmol/l Gly Ala Val Leu Ile Pro Ser Thr Cys Gln Asn NAGase Phe Trp 15 mmol/l 91.7 % Asp Glu Asp > Glu 15 mmol/l His > Lys > Arg 1 Tab. 1 Effects of some amino acids on the enzyme activity (mmol/l) % (mmol/l) % (mmol/l) % Phe His Gly Trp Lys Ala Ser Val Arg Thr Leu Cys Glu Ile Gln Pro Asp Asn Phe Glu Asp NAGase NAGase pnp-β-d-glcnac 1 Asp Glu IC mmol/l Asp Glu 2.3 NAGase Asp Glu Marine Sciences/Vol.30,No.3/

3 1 Glu Asp Fig.1 Effects of Glu and Asp on the enzyme activity 0.5 mmol/l Asp Glu NAGase pnp-β-d-glcnac 2 Asp Lineweaver-Burk Asp 3 Lineweaver-Burk Asp K m Asp V m Asp (S) (E) (ES) Asp (K m ) Asp K I = 9.50 mmol/l 2.5 NAGase Glu Lineweaver-Burk 4 Glu 1/υ 1/c s Glu K m Glu V m Glu NAGase I mmol/l 2 Asp Glu Fig.2 The effect of concentrations of amino acids on the enzyme activity a.l-asp b.l-glu 1~6 0,5,10,15,20 25 mmol/l 1~6 were 0,5,10,15,20 and 25 mmol/l, respectively 2.4 Asp NAGase 3 Asp NAGase Fig.3 Lineweaver-Burk plots for inhibition of Asp on the NAGase from the prawn (Litopenaeus vannamei) 1~ mmol/l, v C s 1~6 were 0,5,10,15,20 and 25 mmol/l,respectively v presents initial velocity of enzyme promotion reaction; Cs presents the concentration of the bottom matters 48 /2006 / 30 / 3

4 4 Glu NAGase Fig.4 Lineweaver-Burk plots for inhibition of Glu on the NAGase from the prawn (Litopenaeus vannamei) 1~ mmol/l v C s 1~6 were 0,5,10,15,20 and 25 mmol/l,respectively v presents initial velocity of enzyme promotion reaction; Cs presents the 3 concentration of the bottom matters NAGase [12~15] NAGase pnp-β-d-glcnac NAGase L L- [16,17] Gly Ala Val Leu Ile Pro Phe Trp Ser Thr Cys Gln Asn Trp NAGase His Lys Arg 15 mmol/l 124.7% 113.7% 109.8% Asp Glu 15 mmol/l 58.1% 74.5%. ph 5.2 -NH 2 H + -COOH Trp Asp Glu ALP [18] [1] Broadway R M, Williams D L, Kaln W C, et al. Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus [J]. Letters in Applied Microbiology : [2] Funke B Spindler K D. Characterization of chitinase from the brine shrimp Artemia [J]. Comp Biochem Physiol, 1989, 94B(4): [3] Lynn K R. Chitinases and chitobiases from the American lobster (Homarus americanus) [J]. Comp Biochem Physiol, 1990, 96B(4): [4] Peters G, Saborowski R, Mentlein R, et al. Isoforms of an N-Acetyl-β-D-glucosaminidase from the Antarctic krill, Euphausia superba: purification and antibody production [J]. Comp Biochem Physiol, 1998, 120B: [5] Peters G, Saborowski R, Buchholz F, et al. Two distinct forms of the chitin-degrading enzyme N-Acetyl-β-D-glucosaminidase in the Antarctic krill: specialists in digestion and moult [J]. Marine Biology, 1999, 134: [6],,,. [J]., 1996, 15(2): [7]. [J]., 2001, 21(2): [8]. [J]. 2001, 27(3): [9] Xie X L, Chen Q X, Lin J C, et al. Purification and some Properties of β-n-acetyl-d-glucosaminidase from Prawn (Penaeus vannamei) [J]. Marine Biology, 2004, 146: Marine Sciences/Vol.30,No.3/

5 [10] Lin J C, Chen Q X, Shi Y, et al. The chemical modification of the essential groups of β-n-acetyl-d-glucosaminidase from Turbo cornutus Solander [J]. IUBMB Life, (9): [11] Chen Q X, Zhang W, Zheng W Z, et al. Kinetic of inhibition of alkaline phosphatase from green crab (Scylla serrata) by N-bromosuccinimide [J]. J Protein Chem, 1996, 15(4): [12] Xie X L, Chen Q X. Inactivation Kinetics of β-n-acetyl-d-glucos aminidase from Prawn (Penaeus vannamei) in the Dioxane Solution [J]. Biochemistry (Moscow), 2004, 69(12): [13],,. N- -β-d-[j]. ( ), 2004, 43(Sup): [14],,,. N--β-D- [J]. ( ), 2004, 43(Sup): [15],,,. N- -β-d-[j]., 2005, 24(1): [16] Fishman W H. On the importance of being stereo specific [J]. Clinica Chimica Acta, 1989, 186(2): [17] Hoylaerts M F, Manes T, Millan J L. Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alka-line phosphatase [J]. Biochem, 1992, 286: [18]. [J]. 1998, 29(4): Inhibitory effects of amino acids on the β-n-acetyl-d-glucosaminidase from prawn Litopenaeus vannamei XIE Xiao-lan 1,2, WANG Yue 1, CHEN Qing-xi 1 (1.The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen , China; 2. Department of Chemistry, Quanzhou Normal College, Quanzhou , China) Received Sep.,20,2004 Key words: Litopenaeus vannamei; β-n-acetyl-d-glucosaminidase; amino acids; inhibitory kinetics Abstract: The β-n-acetyl-d-glucosaminidase (NAGase, EC ) catalyzes the cleavage of N-acetylglucosamine polymers. It is a composition of the chitinases and cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine. It is widely distributed in animal tissues and in microorganisms. The effects of some amino acids on the activity of NAGase from the viscera of prawn Litopenaeus vannamei were studied. The results showed hydrophobic amino acids and neutral hydrophilic amino acids had no effects on the enzyme activity apparently. The alkaline amino acids activated the enzyme activity lightly, and the order of activation was His > Lys > Arg. The acidic amino acids had inhibitory effects on the enzyme activity, and the inhibitory effects were reversible with remaining enzyme activity. The IC 50 s of Asp and Glu (the inhibitor concentrations leading to 50% activity loss) were estimated to be 20 and 28 mmol/l, respectively. The inhibitory mechanisms were competitive types and their K I s were determined to be 9.50 and mmol/l respectively. (: ) 50 /2006 / 30 / 3

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