Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 2, K 3 (A 6D )

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1 ISSN CN gQ 16 2 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 2, (P I4-K) 3 1) (,, ; 1), ) (P I 42K) (A 6D ) 2, (m o lgl ) m o lgl P I42K 50% F ITC,, H el a, (P I 42K) Q 735 D evolopm en t of the M onoclona l An tibody Aga in st Type g Phospha tidylinositol 4-k ina se from Bov ine Cerebella Cortex and its Inh ibitory Effect on Cell Growth YU E J ia2chang,ou YAN G Y ing2sh i, GUO Zhen2quan 1) (N ational L aboratory of B iom acrom olecules, Institute of B iop hy sics,a cadem ia S inica, B eij ing , China; 1) Colleg e of L if e S ciences, P ek ing U niversity, B eij ing , Ch ina) AbstractA m onoclonal an tibody (A 6D ) again st pho sphatidylino sito l 42k inase (P I 42K )w as de2 veloped and characterized. T he resu lts indicated that the b inding affin ity con stan ts of the an tibody to an tigen (P I 42K) either coated o r in so lu tion w ere (m o lgl ) - 1 and (m o lgl ) - 1 re2 spectively. T he F ITC2A 6D con jugated w ith am ph iph ilic p ro tein m icro2vesicu lar w as located m ain2 ly in the p lasm a m em b rane of the H el a cells ob served by fluo rescence m icro scopy. T he P I 42K ac2 tivities cou ld inh ib it 50% by A 6D of m o lgl of cell lysateṡ T he fu rther study show ed that the an tibody cou ld m arkedly inh ib it the grow th of bo th H el a cells and m ou se cerebella granu le cellṡ Key wordsm onoclonal an tibody, Pho sphatidylino sito l 42k inase (P I42K) P I42K,, P I42K (P I42K) (P I) D 4, P IP, P IP (5) (P I42K) K P IP 2; P IP 2 C In s (1, 4, 5) P 3 DA G In s (1, 4, 5) P 3 ; DA G PKC, [ 1 ] 3 ( ) :,, , T el: (010) , Fax: (010) , P I42K E2m ail: Yuejc@ sun5. ibp. ac. cn P I42K, : , :

2 2 : (P I 42K) , [ Χ2 32 P ] (H eparin2sepharo se CL 26B ), A T P (2 000 C igm o l) (D EA E210, FPL C) 95%, P I Sigm a, A T P SERVA, PM SF TL C M erck, SD S2 PA GE B IO 2RAD, Pho s2 phocellu lo se (P211) W hatm an ; H ep2 arin2sepharo se CL 26B Pharm acia, D EA E2 10 FPL C B I02RAD P I24K 3, [2 ] ( ), 15 l (20 mm o lgl T ris2hc l, 10 mm o lgl D T T, 50 m o lgl A T P, 015% T riton X2 100) 25 l 012 m ggm l P I 04, 5, [Χ2 32 P ]A T P (2 C i) 10 mm o lgl M gc l2 50 g, 3 d 50 l20 m in 014 m l 1 m o lgl HC l 016 m l 1500), (21), 20 s, (6 000 rgm in, 2 SP2g0, m in), 014 m l (11) 2%HA T ( 2 2 ),, 20% DM EM,, TL C P IP,, (EL ISA ), 1 nm o l P IP 113 L ow ry [ 3 ], BSA 0101 m o lgl PBS, 0105% ( ) g, g 30 m in, (50 mm o lgl T ris2hc l, 4% T riton X2100, 150 mm o lgl KC l, 2 mm o lgl ED TA, 011 mm o lgl PM SF, 4 mm o lgl Β2, ph 714) 11 (V gv ) ;, P I24K 1 h, g30 m int riton X % 118 W estern blot (N H 4) 2SO 4 1 h, g 30 m in (10 mm o lgl, 011 mm o lg L ED TA, 011% T riton X 2100, 011 mm o lgl 300 m l, 115 m lgm in, 014 m o lgl N ac l 115SD S-PAGE SD S2PA GE L aemm li [ 4 ], 4% 12% 200 V, R 2250 BALB gc (46 ) 100 g (3 ),, 50% (PEG,M r P I24K 1 g, 100 l, ph 916 Tw een220; B io2r ad IgG; (TM B ), 2 m o lgl H 2SO 4, D ynatech m in ireader2g A 450 nm 012,, 34 (A 6D ) BALB gc, 1015 d, (33% ) 1 h, PM SF, 2 mm o lgl Β2, ph 714) 24 h, D EA E210 95% 24 h, : 160 kd, 55 kd, 25 (P211, 5 cm 20 cm ) 0014 m o lgl N ac l kd W estern b lo t [7 ],

3 SD S2PA GE (100 V, 2 h) N C ; A 6D 125 N C 4 66 m o lgl 79 m o lgl ; 15000, NBT BC IP 119 m o lgl 50% ; T riton X2 [ 5 Beathy ] %,W o rtm ann in A m o lgl 50% K aff= (N - P I24K 1) g2{n ( [A b ] - [A b ]) }, 1110 P I42K (g ) [6 ] 212 P I4-K, EL ISA, A 450 nm (2 ) A 6D K lonz K d 1111 kd, 55 kd, 25 kd F ig. 1 [7 ], W estern b lo t A 6D P I42K 4 24 h F ITC A 6D P I42K [8 ] 2A 6D (, m o lg L ) , 5% 56 h, PBS, A 6D 1112 H el a, 2A 6D ( m o lgl ), 37, 6 h, PBS 012% 5 m in, g (10% 1640 ) 37 5 d M T T 213 A6D P I4-K (011% ) 24 h, 7 F ig. 2 A 6D d 37 (10% 1640 ) 24 h,, 011 g 013 g 016 g (P I42K) 2A 6D 6 h PBS 50% A 6D , m o lgl 119, A 6D 3 d M T T P I42K (m o lgl ) A6D P I4-K 2 [ P I ], [A T P ] L inew eaver2burk P I24K P I A T P K m, T riton X2100 W o rtm ann in, 50 P I42K 116, IgG2b SD S2PA GE A 6D 160 F ig. 1W estern blo t of A 6D w ith P I42K L ane 1, Standard p ro teins; lane 2, purified P I42K; lane 3, a part of purified P I42K; lane 4 and 5, W estern blo t: lane 4, Purified P I42K; lane 5, a part of unpurified P I42K 1110, 211P I-4K 0103 g 1 m ggm l 100 A 450 nm 4% T riton X2100,, 5% ( ) SD S2PA GE 95%, A 450 nm A 6D, 56 kd , 18%, A 450 nm A 6D 450 nm o lm g - 1 m in - 1, A 6D 3%

4 2 : (P I 42K) 237 PBS A 6D, 5 d, M T T A 6D, A 6D H el a 15% 319 (N = 8), 20% 118 (N = 6) F ig. 5, : P < 0105, F ig. 2EL ISA curves fo r A 6D at different P I42K2coating amounts T he coating amounts w ere 0. 1 g ( ), 013 g ( ), 016 g ( ) in 100 l; T he antibody at an o riginal concentration of mo lgl w as serially diluted by a facto r of 10 F ig. 3 A 6D [ 9 ] F ig. 4F luo rescence m icro scopy of A 6D 2F ITC in H el a cell (m o lgl ) - 1 Ex: 490 nm; Em; 530 nm F ig. 3A 450 versus concentration of free P I42K in competi2 tion EL ISA fo r A 6D T he concentration of P I42K stock so lution and A 6D are and mo lgl respectively F ig. 5Cell survival determ ined by M T T assay 1. Contro l of H el a cell; 2. H el a cell w ith A 6D 3. Cerebella granule cell; 4. Cerebella granule cell w ith A 6D Survival w as determ ined by M T T assay. D ata are m eans S. E. fo r 8 experim ents 216A6D P I4-K 215A6D A 6D P I42K F ITC, P I42K A 6D 2F ITC 37 A 6D, ( 56 h, A 6D 2F ITC ) F ig. 6 A 6D A 6D 119 (F ig. 4), 10-7 m o lgl 50%

5 F ig. 6Concentration2responce relationsh ip curve of inh ibi2 tion of P I42K by A 6D 1 m ggm l p roetein from H el a cell lysis m em brane w ere incu2 bated w ith varieties A 6D at 4fo r 30 m in after assay,a 6D, 3 A 6D P I42K A T P, A 6D K m 79 m o lgl ; P I K m 66 m o lgl ; P I42K : , W o rtm ann in, m o lgl P I42K 50% 56 kd P I42K A 6D (g ) P I42K P I42K ; P I42, IgG2b, K 160 kd, 56 kd, 24 kd, : (m o lgl ) (m o lgl ) - 1 W estern b lo t A 6D P I42K (References), W estern b lo t P I42K P I42K, m o lgl P I42K 50% ; F ITC,,, P I42K P I P IP, P IP 2 P IP 2, [ 9 ],, P I42K, P I42K 100, ; A 6D P I42K [ 10 ], P I42K [ 11 ] : A 6D P I42K, P I42K 1 Geh rm ann T, L udw ig M G, H eilm eyer T. Pho sphatidylino sito l 42Kinases (Review ). E u r J B iochem, 1998, 253: L ow ry O R, B rough N J, Farr A. P ro tein m easurem ent w ith the fo lin pheno l reagenṫ J B iochem, 1951, 193: L aem li U K. C leavage of structural p ro teins during the assem 2 bly of the need of bacteriophage T. N atu re, 1970, 227: B lake E. A rap id seritrie m ethod fo r detection of alkaline pho s2 phatasre2conjugated anti2body. A nal B iochem, 1984, 136, Beathy J D, Beatty B G, V labo s W G. M easurem ent of mono2 clonal antibody affinity by non2competitive enzym e imm unoas2 say. J Imm un M ethod s, 1987, 100: F riguet B, Challo tte A F, D javadio C. M easurem ent of the ture affinity constants in so lution of antigen2antibody comp lexes by em zym e2linked imm uno so rbent assay. J Imm unol M ethods, 1985, 77:

6 2 : (P I 42K) N airn R C. F luorescence P rotein T racing, 4th ed. Edinburch, London and N ew Yo rk: Churchill L iving Sho re, 1976: 369 8,,,, 1 1, C (W ang X i2de, Zhou Jun2m ei, Guo Zhen2 ( ) (W ang Chong2qin, L i Q i2jing, Guan ying2hui, Peng Gang, Guo ya2qian. The induced acquirem ent of the dono r2specific to lerance to m ice skin allo2 grafts. A cta S ci N atu r U niv P ek inensis), 1998, 34 (4) : Fukam i K M, O sam u N, N ish i E. A ntibody to pho s2 phatidylino sito l 4, 52bispho sphate inhibits oncogene2induced m itogenesis. P roc N atl A cad S ci U SA, 1988, 85: ,,. quan. P reparation and characterization of monoclonal antibodies against adenylate k inase, S ci Ch ina, Series C), 1997, 27 (6) : Glen S, Greg J, Barritt A, F ranceis K. Purification and chem ical modification of a pho sphatidylino sito l kinase from sheep brain. E u r J B iochem, 1991, 201: th FAOBM B Sym po sium Perspectives of B iochem istry and M o lecular B io logy in 21 Century RNA , ( ) ( )