ISSN 100727626 CN 1123870gQ 16 3 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, 352 356 2000 6 (TAFP) 3 1) 1) 1) 3 3 (, 400716; 1), 408435) (TA FP), TA FP, TA FP ph 715, ph 610 TA FP : ST I> an tipain> SBB I> an titryp sin> TL CK> leupep tin > bacteracin> PM SF > T PCK, 1, 102phenan th ro line TA FP TA FP ; ;,, Q 51 Properties of F ibr inolytic Prote in from Tabanus am aenus W a lker (TAFP) and Its Effects on Hem orheology in Ra ts 3 YAN G X ing2yong, YAN Guang2fan 1), HU Kai2zh i 1), ZHAN G Yu2fang 1),LU X iao2feng, PE I Yan 3 3 (B iotechnology Center of S ou thw est A g ricu ltu ral U niversity, Chong qing 400716, Ch ina) ( 1) Institu te of M ed icinal P lant P lantation of Chong qing, Chong qing 408435, Ch ina) Abstract In v itro, on fib rin p late and p lasm inogen2free fib rin p late, the fib rino lytic p ro tein from T abanus am aenus W alker (TA FP ) show ed the sam e fib rino lytic activity as the m ix tu re of TA FP and p lasm inogen indicating that TA FP w as a fib rino lytic enzym e degrading fib rin directly, no t a p lasm inogen activato r, degrading fib rin via activating p lasm inogen. TA FP w as found also to de2 grade fib rinogen. Its op tim um ph w as 7. 5. Inh ib ition of TA FP activity by p ro tease inh ib ito rs show ed: soybean tryp sin inh ib ito r> an tipain> soybean Bowm an2b irk inh ib ito r> an titryp sin> N 2 P2to luenesu lfonyl2l 2lysine ch lo rom ethyl ketone > leupep tin > bacteracin > phenylm ethylsufonyl fluo ride> N 2P2to luenesu lfonyl2l 2phenylan ine ch lo rom ethyl ketone, and 1, 102phenan th ro line had no inh ib ition of TA FP activity. It w as m o st rem arkab le, versu s con tro l, that TA FP cau sed p ro2 longation of b leeding tim e, inh ib ition of p latelet aggregation, decrease of fib rinogen level in p las2 m a, w ho le b lood visco sity, p lasm a visco sity and eryth rocyte sedim en tation, thu s reducing hem al2 ocrit velocity in v ivo. Key words T abanus am aenus W alker, F ib rino lytic p ro tein, P roperty, H em o rheo logy (T abanus biv itta2 tus M artṡ ),, 3 (962103) 3 3 T el: (023) 68251883, Fax: (023) 68864993 E2m ail: peiyan@ sw au. edu. cn, [ 1 ],,,,,, 1965 9,, : 1999206230, : 1999209227 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
3 : (TA FP) 353 (T. am eanusw alker), 116 TAFP :,, TA FP, 2 h, 15 l, 35 l, 0102 m o lgl (ph 714) T ris2hc l ( 0115% Tw een2 [ 2 ] 80) 10 l (ch rom ozym P), 28 (TA FP) 5 m in, M odel 550 405 nm A,, TA FP 117 ph TAFP : J. H. 1 111 :, (30 m g gkg d) (15 m g - 20 112 : Sephadex G275 Pharm acia ; ) 9, 7 d, 1, ( ) 4 h,,, ; AD P 11811 : 015 cm, ch rom ozym P (N 2bu tyloxyoarbonyl2l 2glycyl2 L 2p ro lyl2l 2argin ine p 2n itroan ilide) PM SF (phenyl2 m ethylsufonyl fluo ride) ST I ( soybean tryp sin in2 h ib ito r ) an tipain an titryp sin 1, 102phenan th ro2 line leupep tin TL CK (N 2P2to luenesu lfonyl2l 2ly2 sine ch lo rom ethyl ketone ) T PCK (N 2P2to luene2 su lfonyl2l 2phenylan ine ch lo rom ethyl ketone) SB2 B I(soybean Bowm an2b irk inh ib ito r) Sigm a 56 10 m in, 3 000 rgm in 10 m in, ; tpa B radfo rd [ 7 ], (m ggdl) (N IBSC) 11814 : 37 113 : W istar,, 240 46 g, ( ) ( ) 114 (TAFP) : 11815 : 80 mm, [2 ] 115 TAFP [ 3 11511 : ] 11816 : ( 1%, 015 ggl,, 37 1 h,, 014 U gm l ), 37 16 h, 3 000 rgm in 10 m in,, 10 250 IU gm l tpa (% ) 11512 : 300 l 0102 m o lgl ph 714 50 l (20 ggl ) 50 l, 37 5 m in, (10 U g, [ 4 N o rth rop ], ch rom ozym P, TA FP 118 : 3, gkg d), ( (37 ), 11812 : [ 5 ] (PR P) (PPP) PPP PR P 41 ( 3165) 10 4 gmm, 5 m o lgl AD P,, 5 m in 11813 :, 150 mm, 112 mm 013 mm 2 211 TAFP, TA FP m l) (118 U gm l) 60 l,, tpa 37 : y = 018101+ 011467x, r= 01979 (y, x,, ) TA FP, tpa ;, 5870 tpa IU m g - 1 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
354 16 TA FP, TA FP 212 TAFP TA FP, tpa, (F ig. 1A ), 85 35 m in, (P < 01001), T ab le 1.,,,, tpa TA FP, tpa tpa,, ; TA FP TA FP,, 37 3 m in, (F ig. 1B ), TA FP, ; TA FP,,, ; (P > 0105), tpa,, T ab le 2.,, TA FP Table 1 F ibrino lytic activity fo TA FP on clo t lysis in v itro T reatm ent t (clo t lysis tim e) gm in P TA FP+ P lasm inogen 1416 311 F ig. 1 A nalysis of the fibrino lytic activity of TA FP on fibrin agar p late heated at 85 fo r 0 m in (A ) and 30 m in (B) 11Contro l (buffer) ; 2. TA FP + p lasm inogen; 3. TA FP; 4. tpa + p lasm inogen; 5. tpa, Table 2 A ntith rom bo tic activity of TA FP in v itro TA FP 1511 219 P > 0105 U K+ P lasm inogen 714 116 U K 4512 616 P < 01001 tpa + P lasm inogen 1018 112 T PA 5114 419 P < 01001 n= 5, x ṡ U K: U rok inase; tpa : T issue p lasm inogen activato r N um ber T reatm ent C lo t fo rm ation Result t (clo t lysis tim e) gm in 1 Fg+ TA FP (37, 3 m in) + T b Pg - - 2 Fg+ Pg (37, 3 m in) + T b TA FP + 1416 311 3 Fg+ TA FP (37, 3 m in) + T b - - 4 Fg (37, 3 m in) + T b TA FP + 15. 1 2. 9 5 Fg+ U K (37, 3 m in) + T b Pg + 7. 4 1. 6 6 Fg+ Pg (37, 3 m in) + T b U K + 6. 7 2. 1 7 Fg+ U K (37, 3 m in) + T b + 4512 6. 5 8 Fg (37, 3 m in) + T b U K + 47. 7 5. 8 n= 5, x ṡ Fg: F ibrinogen; T b: T h rom bin; Pg: P lasm inogen; U K: U rok inase; + : C lo t fo rm ation; - : N o clo t fo rm ation 213 TAFP TA FP T ab le 3. ST I an tipain SBB I ; PM SF TA FP ; leupep tin an ti2 TA FP 1, 102phenan th ro line, tryp sin TL CK TA FP ; TA FP ; TA FP T PCK, T PCK 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
3 : (TA FP) 355 1 mm o lgl ( 015 mm o lgl ) TA FP Table 3 Effects of inh ibito rs on TA FP Inhibito r Bacteracin (1 ggl ) ST I(1 ggl ) PM SF (5 mmo lgl ) L eupep tin (1 ggl ) A ntipain (1 ggl ) A ntitryp sin (1 ggl ) Residual activities of TA FP (% ) 4117 414 418 215 5119 1211 3216 1012 611 414 1119 615 F ig. 2 Effects of ph on TA FP activity ph stability; Op tim um ph 1, 102Phenanthro line (1 ggl ) 100 TL CK (1 mmo lgl ) T PCK (1 mmo lgl ) SBB I(1 ggl ) A ctivity w as x s from five rep licates 1213 316 5211 1517 615 311 214 ph TAFP F ig. 2, TA FP ph 715, ph 615 815, TA FP ph 515 1010, ph 610 ( 35416 8717, P < 0101; 26911 6713, P < 0105) ; ( 3614 1113, 4218 1715, P < 0105) ; ( 18011 5419, P < 0101; 2101918 5115, P < 0105), ( 212 013, 214 012, P < 0105) ( 115 011, 116 011, P < 0105) ; 215 TAFP ( 3814 511, 3917 7 d, 517, P < 0105), ( 116 011,, T ab le 4 TA FP 117 013, P < 0105) Table 4 Effects of TA FP on hemo rheo logy of rats Item Do se 30 m g kg - 1 d - 1 15 m g kg - 1 d - 1 0 B leeding tim egs 35416 8717 26911 6713 21317 3618 P latelet aggregation (% ) 614 1113 4218 1715 5311 1412 P lasm a fibrinogen contentgm g dl - 1 ) 18011 5419 21018 5115 26917 6214 W ho le blood visco sity 212 013 214 012 311 013 P lasm a visco sity 115 011 116 011 118 012 ES (% ) 3814 511 3917 517 4318 613 H em alocritgmm h - 1 116 011 117 013 215 111 ES: E ryth rocyte sedim entation. n= 9, x s 216 TA FP, TA FP ;,,, ;,,, [ 6 ], ;,,, 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
356 16, 3,, (Yang X ing2yong, L u X iao2feng, Pei Yan. Purification and characterization component of T abanus quinquecinctus R i2 (References) cardo. A cta E ntom o S in), 1998, 41: 231 236 4 N o rth rop J H, Kunitz R M. C ry stalline enzym e. N ew Yo rk: 1,, (L iu D a2you, Xu Co lum bia U niversity P ress, 1948 L i, L i L i. A dvances in tabanids. Chin T rad itional H erbal 5, : D rug s), 1997, 28: 440 441 (L i Yi2kui, W ang Q ing2m ao ed. P harm acolog ical 2,,,, (T abanus a2 M ethods in Chinese T rad itional D rugṡ Shanghai: Shanghai Sci2 m aenus W alker) ence and T echno logy P ress), 1991: 141 146 (Yang X ing2yong, Cheng J ing2qiu, Pei 6 B radfo rd M. A rap id and sensitive m ethod fo r the quantitation Yan, Yan Guang2fan, Zhang Yu2fang. Purification and bioac2 of m icrogram quantities of p ro tein utilizing the p rincip le of p ro2 tivity assay of the fibrino lytic p ro tein of T abanus am aenus tein dye binding. A nal B iochem, 1976, 72: 243 254 W alkeṙ Ch in J B iochem M ol B iol), 1999, 15 (4) : 580 584 2000 2000 10 24 27 1 : Steven Hou ( N IH ) ( 2) : 13 ) : 11 : 21 31 41 G 51 61 71 81 91T GF2Β W n t 101 ( ) : : : : 13 700, 800 : 2714, : 100080 700 :, E2m ail: isst@b ilong. com 2000 8 31 1) : ) : : : 6510005032650001137 : 100080 :, 8 31 : D ṙ Steven Hou, Investigato r N C I2F rederick Cancer R esearch and D evelop2 m en t Cen ter N IH,M D 21702,U SA T el: 301284621589, Fax: 301284626145 E2m ail: shou@ncifcrf. gov : : 862102826225664, : 862102625322114 W eb: www. ciccsṫ o rg. cngisst (, 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net