Investiga tion on L ipoprote in (a) Receptor in the M em brane of M onkey L iver Cells

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1 ISSN CN gQ 16 1 Ch in J B iochem M o l B io l 2000, V o l 16, N o 1, (a) 3 (, ; 3, ) SD S2PA GE, LDL LDL L p (a) PL G 3, 370 kd 290 kd 80 kd, L p (a) + PL G L p (a) L p (a) L p (a), L p (a) + PL G PL G PL G PL G ; L p (a) + LDL L p (a) L p (a) L p (a), L p (a) + LDL LDL LDL LDL, L p (a) PL G LDL, LDL PL G L p (a) (a),,, Q 51 Investiga tion on L ipoprote in (a) Receptor in the M em brane of M onkey L iver Cells W O X ing2de, TAN G L i2hua, L IBai2hua 3 (M olecu lar M ed ical Institu te of Z hej iang T rad itional Ch inese M ed ical Colleg e, H ang z hou ; 3 B iochem istry S ection of S hanx i T rad itional Ch inese M ed ical Colleg e, T aiy uan ) Abstract To investigate w hether ex ist lipop ro tein (a) recep to r on the m em b rane of m onkey liver cell T he m em b rane p ro tein s from m onkey liver cell w ere analyzed w ith w estern b lo t and EL ISA A fter the m em b rane p ro tein s on the n itrocellu lo se w ere incubated w ith co rresponding an tibody and ligand th ree differen t bands w ere distingu ished T heir m o lecu lar w eigh ts w ere 370 kd, 290 kd and 80 kd respectively E lisa analysis show ed that there w as no m arked difference betw een the m em b rane p ro tein s incubated w ith L p (a) + PL G and w ith L p (a) reacted w ith L p (a) an tibody, bu t it w as m o re sign ifican t if the m em b rane p ro tein s w ere incubated w ith L p (a) + PL G and react2 ed w ith PL G an tibody then if the m em b rane p ro tein s w ere so lely incubated w ith PL G and reacted w ith PL G an tibody L ikew ise, the experim en t show ed that there w ere no clear distinction s be2 tw een the m em b rane p ro tein s that w ere incubated w ith L p (a) + LDL and w ith L p (a) reacted w ith L p (a) an tibody, bu t it w as m o re pow erfu l if the m em b rane p ro tein s w ere in subated w ith L p (a) + LDL and reacted w ith LDL an tibody then if the m em b rane p ro tein s w ere so lely incubated w ith LDL and then reacted w ith LDL an tibody T he experim en t suggests that the m em b rane of m onkey liver cells m ay have the co2ex istence of such th ree differen t recep to rs as L p (a) recep to r, LDL recep to r and p lasm inogen recep to ṙ Key words L ipop ro tein (a), L ow den sity lipop ro tein, P lasm inogen, R ecep to r [ 1 (a) (L p (a) ) Berg 1963 ] Λ (LDL ), apob 2100, L p (a) :,, ,, T el: (0571) , Fax: (0571) E2m ail: woxdf@m ail hz zj cn apo (a), : , :

2 1 : (a) 97 (PL G) [ 2 ] Λ L p (a) 4 10 m in,,, g 4 60 m in,, [ 3, 4 ] Λ L p (a) : 1) 1g4 ( 40 g ) 40 m l B ( 250 L p (a) LDL [ 5 ], mm o lgl T ris2m aleate, 2 mm o lgl CaC l2, 1 mm o lgl LDL, LDL, L p (a), LDL C (10 mm o lgl T ris2m aleate, 2 mm o lgl Ca2 L p (a) 2)L p (a) LDL C l2, 10% T riton X2100, 1 mm o lgl PM SF, ph 610), L p (a) LDL, L p 80 m l, 4 20 m in, g 4 (a) apo (a) apob 2100, 60 m in,, apob m l, - 20, apob 2100 L p (a), SD S2 [ 6, 7 LDL ] 3)L p (a) PA GE,,,, 5%BSA 1 h LDL apo (a),, LDL L p (a), L p (a) LDL PL G 215 h, A (20 mm o lgl [ 8 10 LDL ] T ris, 90 mm o lgl N ac l, ph 714), B ( A 0105% Tw een220), L p (a) 10 m in, A 1 111, 2 h,, 115 h, LDL, PL G ph h, 015% Tw een220,, 110% BSA B io2r ad, 1 h, L p (a) LDL PL G L p (a) LDL L p (a) + PL G, 2 h, L p (a) apob 2100 L p (a) PL G, 115 hλ L p (a) 11019< d< 115 h, ggm l LDL 11060< d < ggm l L p (a) L p (a) Sepharo se 6B L H 2SO 4, 490 nm A, LDL L p (a) SD S2PA GE 2, LDL L p (a) LDL F ig 1 L p (a),,, 370 kd 290 kd 80 kd L p (a) apob 2100, L pb, apob 2100 L p (a) apoa l PL G ; L P (a) + PL G PL G PL G PL G,, 0115 m o lg L N ac l, 162 g 800 m l A (20 mm o lgl T ris2hc l, 0115 m o lgl N ac l, 1 mm o lgl PM SF, ph 610), 2 m in 2, LDL, apob 2100 L p (a) PL G, 3 m o lg F ig 2 L p (a) + PL G L p (a) L p (a) L p (a) F ig 3 L p (a) + LDL L p (a) L p (a) L p (a) PM SF, ph 810), 800 g ; L p (a) + LDL LDL

98 16 LDL LDL F ig 3 A nalysisi of L p (a) and LDL recep to r on liver cell F ig 1 W estern blo t analysis of different recep to rs on monkey liver cell m em brane P ro teins from monkey liver cells

3 98 16 LDL LDL F ig 3 A nalysisi of L p (a) and LDL recep to r on liver cell F ig 1 W estern blo t analysis of different recep to rs on monkey liver cell m em brane P ro teins from monkey liver cells w ere transferred to nitrocellulo se (N C ) after SD S2PA GE T he N C w as cut into several strip s and incubated w ith different lig2 ands and antibodies 1: P ro tein mo lecular w eight standard; 2: N C w as incubated w ith LDL recep to r antibody derived from bovine adrenal co rtex; 3: N C w as incubated w ith LDL and then w ith apob2100 anti2 body; 4: N C w as incubated w ith L p (a) and then w ith L p (a) antibody; 5: N C w as incubated w ith PL G and then w ith PL G antibody m em brane from monkey w ith EL ISA T he m em brane p ro tein from monkey liver cell w as coated A fter block ing w ith 1% BSA 2 Λggm l LDL and 011 m ggm l L p (a) w as added in increasing dilution from 1 5 to A fter w ashing co rresponding antibody w as app lied T he reaction w as stopped w ith 3 mo lgl H 2 SO 4 and w as m easured at 490 nm L P (a) + L p (a) antibody; LDL + LDL antibody; (L P (a) + LDL ) + L p ( a) antibody; (Lp (a) + LDL ) antibody F ig 4 T he interaction of L p (a) and PL G w ith their anti2 F ig 2 A nalysis of L p (a) and P lasm inogen recep to r on liv2 er cell m em brane from monkey w ith EL ISA T he m em brane p ro tein from monkey liver cell w as coated A fter block ing w ith 1%BSA 2 Λggm l PL G and 011 m ggm l L p (a) w as added in increasing dilution from 1 5 to A fter w ashing co rresponding antibody w as app lied T he reaction w as stopped w ith 3 mo lgl H 2SO 4 and w as m easured at 490 nm L p (a) + L p (a) antibody; L PG+ PL G antibody; (L P ( a) + PL G) + L p ( a) antibody; (L p (a) + PL G) + PL G antibody bodies T he 2 Λggm l PL G and 011 m ggm l L p (a) w as coated in in2 creasing dilution from 1 5 to A fter w ash ing co rre2 sponding antibody w as app lied The reaction w as stopped w ith 3 mo lgl H 2SO 4 and w as m easured at 490 nm L p (a) + PL G antibody; L p (a) + L p (a) antibody; PL G+ L p (a) antibody; PL G + PL G antibody F ig 4 PL G L p (a), L p (a) PL G F ig 5 apob 2100 L p (a), L p (a) LDL

4 1 : (a) 99 F ig 5 T he interaction of L p (a) and LDL w ith their anti2 bodies T he 2 Λggm l LDL and 011 m ggm l L p (a) w as coated in in2 creasing dilution from 1 5 to A fter w ash ing co r2 responding antibody w as app lied The reaction w as stopped w ith 3 mo lgl H 2SO 4 and w as m easured at 490 nm L P (a) + L p (a) antibody; L p (a) + LDL antibody; LDL + L P (a) antibody; LDL + LDL antibody L p (a),, 3 LDL L p (a), L p (a) L p (a), LDL ( F ig 5) L p (a) [ 11, 12 ] L p (a),, LDL PL G L p,,, (a) SD S2PA GE, (References),, 3, LDL LDL, 290 kd (, LDL ), L p (a), 370 kd, PL G, 80 kd,, L p (a) + PL G L p (a) L p (a) L p (a), L p (a) + PL G PL G ( F ig 2) PL G L p (a), PL G L p (a) PL G L p (a), L p (a), L p (a) L p (a), PL G PL G ; PL G L p (a),, , L p (a), PL G, L p (a) + PL G PL G PL G PL G, PL G L p (a), L p (a) K ringle g, PL G PL G L p (a), L p (a) PL G,, PL G L p (a), L p (a) PL G ( F ig 4), L p (a) LDL + L p (a) L p (a) apob 2100, L p (a) + LDL L p (a) L p (a) L p (a), L p (a) + LDL LDL ( F ig 3), L p (a) LDL L p (a) LDL, LDL 1 Berg K A new serum type system in m an: L p system A cta p athol M icrobiol S cand 1963, 59: Berg K Impact of m edical genetics on research and p ractices in the area of cardiovascular disease 312 C lin Gene, 1989, 36: Scandcamp M, Simon D İ L ipop ro tein (a) is an independent risk facto r fo r m yocardial infraction at a young age C lin Chem, 1990, 36: Zenker G, Ko tringer P, Bon G, N iederko rn K, Pfeiffer K, Ju2 gens G L ipop ro tein (a) as a strong indicato r fo r cerebrovas2cu2 lar disease S troke, 1986, 17: U term ann G, Hopp ich ler F, D iep linger H, Seed M, T homp son G, Boerw ink le E D efects in the LDL 2recep to r gene affect L p (a) levels: m ultip licative interaction of two gene loci associated w ith p rem ature arterio sclero sis P roc N atl A cad S ci U SA, 1989, 86: Steyrer E, Ko stener GM Interaction of lipop ro tein L p (a) w ith the BgE2recep to r: a study using iso lated bovine adrenal co rtex and hum an fibroblast recep to rs J L ip id R es, 1990, 31 ( 7) :

5 W illiam s K J, F less G M, Petrie K A, Snyder M L, B rocia R W, Sw enson T L M echanism by w hich lipop ro tein lipase al2 ters cellular m etabo lism of lipop ro tein (a), low density lipop ro2 tein, and nascent lipop ro teins Ro les fo r low density lipop ro tein recep to rs and heparan sulfate p ro teoglycans J B iol Chem, 1992, 267 (19) : Kremp ler F, Ko stner G M, Ro scher A, H aslauer F, Bo lzano K, Scandhofer F Studies on the ro le of specific cell surface re2 cep to rs in the removal of lipop ro tein (a) in m an J C lin Invest, 1983, 71: M artm ann2m oe K, Berg K L p (a) lipop ro tein enters cultured fibroblasts independently of the p lasm a m em brane low density lipop ro tein recep to ṙ C lin Genet, 1981, 20: Spandrio S, Sleim an I, Scalvini T, Salvi A, D i Stefann O, Pagliaini R, Balestrieri G P L ipop ro tein (a) in thyro id dysfunc2 tion befo re and after treatm enṫ H orm M etab R es, 1993, 25 (11) : M iles L A, Gunther M F, L evin E G, Scanu A M, Edw ard F, P low A p ro tein basis fo r the th rom bo tic risk s associated w ith lipop ro tein (a) N atu re, 1989, 339 (6222) : Edelberg M J, M ario G G, Salvato re V P lipop ro tein a inh ibits strep tokinase2m ediated activation of hum an p lasm inogen B io2 chem istry, 1989, 28: , 40, 180, Λ, Λ Λ Λ Λ, Λ 1 4,, ; 5 9 Λ,,, ; 10,,,, Λ,, Λ,, Λ,, Λ, 158, Λ : : (010)

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