kd Km = ^ Eqn 2-6.
So if kd >> kdiff then = kdiff
In th is case the gradient o f a p lo t of 1 /k vs [M+] w ill have a g ra d ie n t o f 1/kd iff. Assum ing th a t th is is the case fo r potassium
tra n s p o rt and th a t the d iffu sio n constants are the same fo r the sodium and potassium complexes gives a value o f 1.5 x lO '^s'i for kdiff w hich can be substituted into equation 2-9 such th a t:
Slope = Eqn 2-12.
kd kdiff
andinte rc e p t = kf.kd .ka ^f ^ -IS . The firs t of these enables a value to be obtained for kd.
"
(slope-(l/kdiff))
2-14
w h ich can then be substitu te d in to the second equation to calculate a value for kf.
k f = i n t r l e p t ' t f f ^ -1 5
m atch the valu e o f Kg obtained graph ically. In th is case the calculated values are given below in Table 2-8.
Table 2-8. The calculated and actual values o f Kg for Na+. k f (lO ^ s -i) kd° KsC (M-1) Ks® (M-1) S alinom ycin 5.74 1.19 4.8 6.1 N arasin 13.32 1.66 8.02 11.0
The values o f Kg are ro u g h ly tw o th ird s o f those expected. T h u s im p ly in g th a t one o f the assum ptions was in v a lid . I t is p rob a b ly in c o rre ct to assum e th a t the p o ta ssium tra n p o rt is com pletely governed b y diffusion, b ut the values are close enough to m ake a diffusion co n trib utio n to the gradient a possibility.
The th e rm o d y n a m ic s tab ility o f th e m eta l/io n o p h o re com plex does n o t have a great effect on tra n sp o rt in a system like ours. The tra n s p o rt rates here are determ ined b y th e k in e tic param eters kf, k^ and k^iff. The s tab ility constant o f the com plex is derived from the ra tio o f k f and k^ b ut it is only o f significance in tra n s p o rt if the com plex is so stable th a t the ca tio n is never released or so unstable th a t the com plex is never form ed. In a com petitio n experim ent, however, the s tab ility o f the com plex can be o f key im portance. If a lo t of the ionophore is sequestered in the m em brane as a stable com plex of one of the com peting ions it can not also be tra n sp o rtin g an io n of the other type. T his m eans th a t the con centratio n o f ionophore involved in tra n s p o rtin g the
m o re s tab le co m p le x w ill be h ig h and th e m ore ra p id ly
tra n sp o rte d io n w ill o n ly have a low con centratio n o f ionophore w ith w h ic h to complex.
2.5.3 The effect o f s tru c tu ra l changes on the tra n s p o rt ab ilitie s o f salinom ycin.
M iyazaki et al had published a study o f the effect o f s tru c tu ra l
m o d ific atio n on the tra n s p o rt and a n tim ic ro b ia l p ro p e rtie s o f
s a l i n o m y c i n .T his was an attem pt to see how changes affected
the tra n s p o rt as seen in o u r system. Some tra n s p o rt studies were
m ade on d ih yd rosa lin om ycin and it was also attem pted to m ake C(20) keto salinom ycin.
These com pounds were prepared from the so d ium s a lt o f s a lin om y c in us in g th e m eth o d s o f A s u k ab e et a l.^ 9 D ih y d ro s a lin om y c in (DSL) was prepared b y h y d ro g e n atio n o f salinom ycin over a pa lladium -carbon catalyst. The p repa ratio n o f 20 -keto sa lin om ycin was attem pted u n su cce ssfu lly b y o x id atio n w ith p y rid in ium chlorochrom ate. The sam ples were id e n tifie d u sin g HPTLC and nm r. There was also only one spot in the TLC o f the sam ple o f dih ydrosa lin om ycin . This and th e loss o f the d istin ctive vin y lic protons in salinom ycin showed th a t re d u ctio n of the double bond had been successful.
A n um b e r o f studies in p h o sp h a tid ylch o lin e vesicles were attem pted, using both 23]vja and 39k nm r. These used the dynam ic lin e broadening experim ent above. O nly one o f these studies, a t 200 m M sod ium concentration, was successful. The o th ers a ll showed in itia l broad lines. T his m eant th a t there w as e ith e r no fu r th e r lin e b ro a d e n in g seen, o r the lin e s m erged before a
s u ffic ie n t n um b e r o f experim ents could be carried out. The data obtained was a value fo r the rate o f efflux at 200m M sodium . T h is
is given in Table 2-9 w here it is com pared w ith th a t seen fo r salinom ycin under the same conditions.
Table 2-9. C om parison betwen the tra n sp o rt rates o f salinom ycin
D ihydrosalinom ycin S alinom ycin
Rate o f efflux (s“ ^) 6.145 ± 0.200 11.310 ± 0.198 F rom these data it can be seen th a t d ih y d ro s a lin om y c in
tra n sp o rts sodium ions at about h a lf the rate of salinom ycin. This agrees w ith the p reviou sly publishe d work^Q th a t re d u cin g the vin ylic fu n ctio n decreases the a ctivity of salinom ycin.
2.6 L ith ium tra n sp o rt w ith salinom vcin and narasin.
U sing m a g n etisa tio n tra n s fe r and ^ L i/ ^ L i exchange it is possible to stu d y the rate o f ionophore m ediated lith ium tra n s p o rt th ro u g h phospholipid bilayers. This is a p re lim in a iy stu d y of the rates o f tra n sp o rt o f Li+ m ediated by salinom ycin and n a rasin in ph osphatidylcholine vesicles.
2.6.1 A com parison between m agnetisation tra n sfe r and isotope exchange.
The aim o f th is w o rk was to measure the tra n s p o rt rates and calcu la te s tab ility , fo rm atio n and dissociation com plexes o f the lith ium complexes w ith salinom ycin and narasin as had been done fo r th e so d ium an d p ota ssium c o m p l e x e s .38 We were also
in te re s te d in th e com p a riso n o f the ra te s obta in e d u s in g
m a gnetisation and isotope exchange experim ents. W ould these
procedures, the fo rm e r an exchange o f n u c le i w ith d iffe re n t
m agnetisations and the la tte r an exchange o f nu clei w ith diffe ren t
masses produce the same results?
The com pariso n o f m a g n etisa tio n tra n s fe r w ith isotope exchange was carried o u t at lOOmM [Li+] w ith n a ra sin as the ionophore. The vesicles were grow n u sin g the same m ethod as given above and the experim ents were carried o u t at 25°C as usual. The re sults are given in Table 2-10.
Table 2-10. C om paring the values obtained fo r n a ra sin m ediated lith ium tra n sp o rt jy different m ethods.
E x p e rim e nt M agnetisation transfer Isotope exchange Rate c o n sta n t/s"i 77.1 ± 0 .9 9 19.96 ± 0.69
There is a qu alitative agreement between the rate constants obtained b ut the values are seen to differ by a fa cto r o f four, the isotope exchange p ro d u cin g the m ore ra p id rate. There are im p o rta n t differences in the experim ental procedure w h ic h m ay account for th is deviation. One is th a t a different set o f vesicles are used fo r each m easurem ent w hen using isotope exchange as the
m ethod of study. In m easurem ents by m agnetisation tra n sfe r only one set o f vesicles are used. T his means th a t there is an extra p o ssib ility of error in the form er m ethod. If the vesicles are n o t a ll the same size, w h ic h was the case here, or have d iffe re n t size d is trib utio n s the n the rates w ill be different. T h is reduces the co rre la tio n between experim ents ru n on d iffe re n t sets o f vesicles and exp la in s the m u ch la rg e r percentage e rro r seen fo r the isotope exchange experim ents. The other proble m lies in the difference in ionophore concentration used. There was a 200 fold
difference in the ionophore/P C ra tio used in the tw o experim ents, w ith the isotope exchange the more dilute. T his w ou ld m a gnify any errors in the p repa ratio n o f the ionophore so lu tio n and its a d d itio n to the experim ental m edium . The data fo r the isotope exchange takes in to account the passive tra n sp o rt o f lith ium across the vesicle m em branes. T h is was m easured b y ru n n in g an experim ent w ith no ionophore added and was seen to have a rate o f 2.25x10‘ 3s"h
2.6.2 A co m p a riso n o f s a lin om v c in and n a ras in to o th e r n a tu ra llv occuring polvether antibiotics.
M a gne tisation tra n sfe r experim ents were carrie d o u t a t a range o f cation concentrations to try to determ ine the s tab ility , disso cia tion and fo rm atio n constants o f the ionophore / lith ium complexes. This was attem pted for both salinom ycin and n a rasin
b ut u n fo rtu n a te ly the data were in su fficie n tly reproducible to allow values for these constants to be obtained. The data was su fficie n tly good, however, to give some idea of the rate and com pare it to the kn o w n values fo r M139603 (see Figure 2 -1 6),^6 m o n e n s i n , ' ^ ^
n ig e r ic in ^ i and some s y n th e tic ionophores w h ic h had been
studied by other m embers o f the g r o u p .3 2 . 5 3 The tra n s p o rt rates
are given in Table 2-11. From these data it is possible to see th a t salinom ycin and narasin both give rates in the same region (20-80
s -i) w ith salinom ycin possibly the faster. This w ou ld be borne o ut
b y the d iffic u lty we, and Caughey et a l,34 fo u n d in p re p a rin g a stable sam ple of lith ium narasin for stud y by 2D nm r, see chapter 3. The tra n sp o rt data is in su fficie n tly good to be sure.
Me Me Me Me, OMe OR Me M 1 3 9 6 0 3 Me
Figure 2-16. The stru ctu re of M139603.
It is obvious however th a t the tra n sp o rt rates fo r lith ium are a lot slower th a n those seen for sodium and potassium . There is a difference o f ro u g h ly three orders o f m agnitude in the observed rates. T h is is as w ould be expected from the previous studies on sa lin om ycin and n a rasin w h ich have a ll observed a m u ch low er lith ium preference fo r these m aterials. This is the firs t s tu d y in p h o sp h o lip id m em branes to dem onstrate lith ium tra n s p o rt w ith eith e r o f these two ionophores.
T able 2 -1 1 . The rate c o n s ta n t fo r e fflu x a t lOOm M c atio n co n centratio n.
LiSL LiNS NaSL NaNS KSL KN S
k ’(s-i) 31.7 19.96 12010 2 9 1 0 0 10 680 0 9 2 6 3 0 It is in te res tin g to com pare these re s u lts w ith tho se obtaine d w ith a n um b e r o f d iffe re nt ionophores w ith the same
system . The firs t type o f ionophore to consider is the n a tu ra lly o ccu rrin g polyether an tibiotics, three of w h ich have been stud ied u sin g o u r system nam ely M l39603, m onensin and n ig e ricin . O f
these M l 39603 and m onensin are sodium selective ionophores and the other is potassium selective. The observed tra n s p o rt rates fo r lith ium show, as m ig ht be expected th a t the form er p a ir are