13
BUx'himica e" Biophysica Acta. 1113 (ITS2) 13-23 ,~". 1992 El~vier ~ i e n c e Puhlisher~ B.V. All rights re.,~:rvcd 0304-4157/q2/$llS.iX)
BBARI-V 853t)7
Intestinal uptake and translnembrane transport systems of intact GSH; characteristics and possible biological role Maria Teresa Vincenzini, Fabio Favilli and Teresa |antomasi l)epartm('nt o[ Bicx'hemical Sciem'es. Uni~'er.~ity. f Florence. Florence (Itah') (Received 14 May ItJql) ( Revi.~d man u~cript received 29 Oct~d~r I ' ~ I )
Contents 1.
Intn~Juction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
II.
GS11 uptake systems in inteslinal muco,,al cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
III.
Findings ab1 and i~ the presence of IINI m M NaS('N to). G S I I uptake values were determined af:er 60 rain of incubation On the a l ~ c i s ~ are reported the =atios of osmolaril.~ inside/osmolarit) outside the ve~icles. The y-inlcrcepl, which i~ greater than (I. show~ that G S H binding to Ihc vcsicle~ is he;• in the presence of Na " and 23r~ in its absent," 135].
have already been characterized. Wc believe that its binding sites on the transport system are mainly el,.ctrostat;c in nature and that the pH can ch.':nge the
'
Addition
/
c 3o
~
I"i/
T I
Jl
~O
~
o.
60
120
GSH laM
5
/ 5'0
100 ~ , ~ laM
Fi$, 6. (At Effect of varying GSII concenlrations on the initial rate of GSH uptake. GSII uptake values w e r e determined al room temperature after l0 s of incubation with ATt: s Ireatcd membrane vesicles (.'~) #I) in uptake media containing: I[I0 mM mannitoL l(i mM Hcpes-Tris (pH 7). l(g) mM NaSCN and varying concentrations of [ ~H)GSI! (14-150 taM). The dashed ',inc is a graphical: ~stimate of the non-saturable component of GSI! uptake. The linear relation betwe,'n C;SH up=ake and initial incubation times (5-20 s) is sh~'n in the upper plot. Experimental conditkms are the ~me" the GSH concentration in uptake media u,as 50 # M . (13) Initial rates ol Iran~l.x)rt-medialcd GSII uptake. The data were obtained by suhstracting the diffusion comw.)nent for each concentration from the oUal GSII uptake {35].
l0 much lower than K m values for facilitated transport systems of other peptides [57]. It is aim evident that these cations do not vary GSH affinity, while they do increase transport capacity. V=,,~ values being 6-fold higher than in the presence of choline. However. the action mechanism of these cations seems to be rather complex. Their effect is .selective and catalytic but not energetic.
V-E. Effect of membrane potential
I ABLI- IV
Effect of .~tnt',tutullv re]at('d comlxmnd~ tm (;SII uptake Tran,,l~rt was carried out with BBMV (311 pl) in uptake media containing ItMI mM mannitol. Iq "~M Hepes-Tri,, (pll 7.5| IIM)mM NaCI. 511 v.M I~IIKiSll arid in the presence of 5IMI , M unlabeled Coml~unds. The data are the means 4-_S.D. of four experiments. The values in parentheses are the inhibition percentages of (;Sit uptake c
BUx'himica e" Biophysica Acta. 1113 (ITS2) 13-23 ,~". 1992 El~vier ~ i e n c e Puhlisher~ B.V. All rights re.,~:rvcd 0304-4157/q2/$llS.iX)
BBARI-V 853t)7
Intestinal uptake and translnembrane transport systems of intact GSH; characteristics and possible biological role Maria Teresa Vincenzini, Fabio Favilli and Teresa |antomasi l)epartm('nt o[ Bicx'hemical Sciem'es. Uni~'er.~ity. f Florence. Florence (Itah') (Received 14 May ItJql) ( Revi.~d man u~cript received 29 Oct~d~r I ' ~ I )
Contents 1.
Intn~Juction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
II.
GS11 uptake systems in inteslinal muco,,al cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
III.
Findings ab1 and i~ the presence of IINI m M NaS('N to). G S I I uptake values were determined af:er 60 rain of incubation On the a l ~ c i s ~ are reported the =atios of osmolaril.~ inside/osmolarit) outside the ve~icles. The y-inlcrcepl, which i~ greater than (I. show~ that G S H binding to Ihc vcsicle~ is he;• in the presence of Na " and 23r~ in its absent," 135].
have already been characterized. Wc believe that its binding sites on the transport system are mainly el,.ctrostat;c in nature and that the pH can ch.':nge the
'
Addition
/
c 3o
~
I"i/
T I
Jl
~O
~
o.
60
120
GSH laM
5
/ 5'0
100 ~ , ~ laM
Fi$, 6. (At Effect of varying GSII concenlrations on the initial rate of GSH uptake. GSII uptake values w e r e determined al room temperature after l0 s of incubation with ATt: s Ireatcd membrane vesicles (.'~) #I) in uptake media containing: I[I0 mM mannitoL l(i mM Hcpes-Tris (pH 7). l(g) mM NaSCN and varying concentrations of [ ~H)GSI! (14-150 taM). The dashed ',inc is a graphical: ~stimate of the non-saturable component of GSI! uptake. The linear relation betwe,'n C;SH up=ake and initial incubation times (5-20 s) is sh~'n in the upper plot. Experimental conditkms are the ~me" the GSH concentration in uptake media u,as 50 # M . (13) Initial rates ol Iran~l.x)rt-medialcd GSII uptake. The data were obtained by suhstracting the diffusion comw.)nent for each concentration from the oUal GSII uptake {35].
l0 much lower than K m values for facilitated transport systems of other peptides [57]. It is aim evident that these cations do not vary GSH affinity, while they do increase transport capacity. V=,,~ values being 6-fold higher than in the presence of choline. However. the action mechanism of these cations seems to be rather complex. Their effect is .selective and catalytic but not energetic.
V-E. Effect of membrane potential
I ABLI- IV
Effect of .~tnt',tutullv re]at('d comlxmnd~ tm (;SII uptake Tran,,l~rt was carried out with BBMV (311 pl) in uptake media containing ItMI mM mannitol. Iq "~M Hepes-Tri,, (pll 7.5| IIM)mM NaCI. 511 v.M I~IIKiSll arid in the presence of 5IMI , M unlabeled Coml~unds. The data are the means 4-_S.D. of four experiments. The values in parentheses are the inhibition percentages of (;Sit uptake c
