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A comparative study of adenylate cyclase activity stimulated by native VIP or different species of monoiodinated VIP
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A COMPARATIVE STUDY OF A D E N Y L A T E CYCLASE ACTIVITY BY NATIVE VIP OR DIFFERENT SPECIES OF MONO[ODINATED VIP.
STIMULATED
J.A. MARIE, D. HUI BON HOA, R. JACKSON* and (3. ROSSELIN, Unite INSERM U.55, Hopital St. Antoine, 18/4, rue du Fg. St. Antoine, 75571 - Paris Cedex 12, *Amersham International Plc, White Lion Road, Amersham, Bucks, HP7 9LL, UK.
Three major forms of monoiodinated vasoactive intestinal peptide (M1251 VIP) were isolated a f t e r chloramine T iodination and HPLC purification. VIP contains a tyrosine resid~e~at positions 1Q and 22. The iodinated tyrosine residue was located in each form of M - - J I - V I P using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. A f t e r which, the HPLC isolated iodinated fragments obtained were used for HPLC comigration studies w i t h iodinated synthetic C anc~ ~'.~ t~_rminal VIP and for amino acid analysis. The first two eluting I:~#~s.~oand 2 are ( M ' ~ - ~ U T y r - V l P ) ; peak 1 has an oxidized r { ~ h i o n i n e ; peak 3 is a (M±=>I-LLTyr-VIP). The a b i l i t y of the d i f f e r e n t species of M ' " ~ I - V I P to stimulate cAMP production in transformed coloni~2Gells in culture (HT-29) was compared to that of native VlP. The potencies of the M I-VIP species expressed as a percentage r e l a t i v e to the potency of native VIP were, peak (2L2: 96, (2): 93; (3): 136, in the range of concentrations tested (2250 pM). The M125 I- Tyr-VIP is significantly more active than native BIP (p> O.Ol). We conclude that although iodination at e i t h e r position increases the hydrophobicity of VIP, only iodinated tyrosine 22 in the apolar helic COOH terminal of VIP is involved in increasing the effectiveness of the VIP receptor interaction. The oxidation state of methionine residue 17 appears unimportant for the stimulation of cAMP production.
H P L C C O N T R O L O F VIP P R O C E S S I N G IN H U M A N T R A N S F O R M E D COLONIC E P I T H E L I A L C E L L S (HT-29) IN C U L T U R E J.C. MARIE, D. HUI BON HOA, (3. HEJBLUM, C. BOISSARD and (3. ROSSELIN, Unite INSERM U.55, Hopital Saint-Antoine, 18/4 rue due Fabourg Saint-Antoine, 75571, Paris Cedex 12.
Vasoactive intestinal Peptide (VIP) possesses specific receptors in gut epithelial ceils which are coupled w i t h adenytate cyclase. U l t r a s t r u c t u r a l proof of internalization of VIP in HT-29 cells ~ a ~ recently been obtained in our laboratory. We have used monoiodinated VIP ( M ' ~ - V I P ) and HPLC analysis with binding studies of the eluate in order to monitor the biochemical characteristics of the internalized ]igand. A f t e r ] h r at O . . . . . . 10 C maximum bmdl~n.of the r a d l o a c t w l t y on the cell membrane was observed and verlfLed to be pure M I~-sVIP. A f t e r which, lnternahzatlon was performed at 37 C for d i f f e r e n t times. The M I-VIP bound to suface receptors was removed by acetic acid and the remaining internalized VIP was e x t r a c t e d a f t e r boiling and sonification of the cells. VIP i n i t i a l ] y b o u n d to cell membrane is (1) internalized m a x i m a l l y before 10 rain; (2) dissociated in the supernatant and a f t e r 60 rain there is no more VIP at the cell su{'~ce. A f t e r 10 and 60 rain of incubation, the percentage of the t o t a l interna~z/~d M - ' - I - V I P which remains unaltered is 44 and 0%, respectively. The unaltered M - ' ~ I V[P has the same retentlb:~ t i m e on HPLC chromatography and binds to rat liver memberane as control M - - ' I - V [ P . We conclude t h a t (1) VIP proceeds rapidly into the cell a f t e r i n i t i a l binding; (2) part of the internalized VIP is still under a biologically active form; (3) internalization of VIP is temperature-and time-dependent; (4) HPLC can isolate unaltered VIP as well as products of processed VIP afb,r internalization in HT-29 cells. •
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A COMPARATIVE STUDY OF A D E N Y L A T E CYCLASE ACTIVITY BY NATIVE VIP OR DIFFERENT SPECIES OF MONO[ODINATED VIP.
STIMULATED
J.A. MARIE, D. HUI BON HOA, R. JACKSON* and (3. ROSSELIN, Unite INSERM U.55, Hopital St. Antoine, 18/4, rue du Fg. St. Antoine, 75571 - Paris Cedex 12, *Amersham International Plc, White Lion Road, Amersham, Bucks, HP7 9LL, UK.
Three major forms of monoiodinated vasoactive intestinal peptide (M1251 VIP) were isolated a f t e r chloramine T iodination and HPLC purification. VIP contains a tyrosine resid~e~at positions 1Q and 22. The iodinated tyrosine residue was located in each form of M - - J I - V I P using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. A f t e r which, the HPLC isolated iodinated fragments obtained were used for HPLC comigration studies w i t h iodinated synthetic C anc~ ~'.~ t~_rminal VIP and for amino acid analysis. The first two eluting I:~#~s.~oand 2 are ( M ' ~ - ~ U T y r - V l P ) ; peak 1 has an oxidized r { ~ h i o n i n e ; peak 3 is a (M±=>I-LLTyr-VIP). The a b i l i t y of the d i f f e r e n t species of M ' " ~ I - V I P to stimulate cAMP production in transformed coloni~2Gells in culture (HT-29) was compared to that of native VlP. The potencies of the M I-VIP species expressed as a percentage r e l a t i v e to the potency of native VIP were, peak (2L2: 96, (2): 93; (3): 136, in the range of concentrations tested (2250 pM). The M125 I- Tyr-VIP is significantly more active than native BIP (p> O.Ol). We conclude that although iodination at e i t h e r position increases the hydrophobicity of VIP, only iodinated tyrosine 22 in the apolar helic COOH terminal of VIP is involved in increasing the effectiveness of the VIP receptor interaction. The oxidation state of methionine residue 17 appears unimportant for the stimulation of cAMP production.
H P L C C O N T R O L O F VIP P R O C E S S I N G IN H U M A N T R A N S F O R M E D COLONIC E P I T H E L I A L C E L L S (HT-29) IN C U L T U R E J.C. MARIE, D. HUI BON HOA, (3. HEJBLUM, C. BOISSARD and (3. ROSSELIN, Unite INSERM U.55, Hopital Saint-Antoine, 18/4 rue due Fabourg Saint-Antoine, 75571, Paris Cedex 12.
Vasoactive intestinal Peptide (VIP) possesses specific receptors in gut epithelial ceils which are coupled w i t h adenytate cyclase. U l t r a s t r u c t u r a l proof of internalization of VIP in HT-29 cells ~ a ~ recently been obtained in our laboratory. We have used monoiodinated VIP ( M ' ~ - V I P ) and HPLC analysis with binding studies of the eluate in order to monitor the biochemical characteristics of the internalized ]igand. A f t e r ] h r at O . . . . . . 10 C maximum bmdl~n.of the r a d l o a c t w l t y on the cell membrane was observed and verlfLed to be pure M I~-sVIP. A f t e r which, lnternahzatlon was performed at 37 C for d i f f e r e n t times. The M I-VIP bound to suface receptors was removed by acetic acid and the remaining internalized VIP was e x t r a c t e d a f t e r boiling and sonification of the cells. VIP i n i t i a l ] y b o u n d to cell membrane is (1) internalized m a x i m a l l y before 10 rain; (2) dissociated in the supernatant and a f t e r 60 rain there is no more VIP at the cell su{'~ce. A f t e r 10 and 60 rain of incubation, the percentage of the t o t a l interna~z/~d M - ' - I - V I P which remains unaltered is 44 and 0%, respectively. The unaltered M - ' ~ I V[P has the same retentlb:~ t i m e on HPLC chromatography and binds to rat liver memberane as control M - - ' I - V [ P . We conclude t h a t (1) VIP proceeds rapidly into the cell a f t e r i n i t i a l binding; (2) part of the internalized VIP is still under a biologically active form; (3) internalization of VIP is temperature-and time-dependent; (4) HPLC can isolate unaltered VIP as well as products of processed VIP afb,r internalization in HT-29 cells. •
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I Z >
.
.
.
.
o