La denominada objeción de conciencia impropia

These antagonists have been designed to prevent the binding o f spermine to the

NMDA receptor channel. They were tested to determine whether they affected enzyme

activity, in which case it could be that there are some structural similarities between the

spermine binding sites on the NMDA receptor channel and methionine synthase.

Concentration % stimulation of Concentration of % stimulation of B12- of arcaine pM B12-MS by arcaine ifenprodil pM MS by ifenprodil .......6... - 23 ± 3* .... ... 2 ... 36 ± 60

62 -12± 11 21 - 3 6 ± 12

617 21 ± 3 6 208 - 26 ± 20

Table 5.3 The effect of arcaine and ifenprodil upon methionine synthase activity.

The e x p e rim e n t w a s p e r f o r m e d in 5 0 m M h e p e s bu ffer p H 7.2 a t 3 7 X! f o r 3 0 m inutes. 3 0 p g o f p r o te in

w e r e u s e d in e a c h a s s a y . A ll v a lu e s a r e m ea n s ± SD , * d e n o te s th e o n ly sig n ific a n t r e s u lt fr o m a tw o s a m p le d t- te s t c o m p a r in g re s u lts to c o n tr o ls (0% ); 0 .0 0 K P < 0 .0 1 , n = 2 f o r a ll values.

Table 5.2. shows that neither compound had a significant concentration-

dependent effect upon enzyme activity, both inhibition and stimulation o f activity was

recorded.. However, it was possible that the compounds bound to the enzyme without

changing activity. To assess whether this was true the highest concentration used in the

previous experiment was incubated with a low concentration (3 pM) o f spermine. This

concentration was chosen so that any interference in binding by the antagonists should

be easily seen, but the concentration was still great enough to cause stimulation of enzyme activity. 110 —1 100 M 90 - M 80 OQ 70 0 60 1 50 - I 40 i 30 ^ 20 - 10 0

■ sperm ine . ® sperm ine + ifen p rod il □ sperm ine + arcaine

Figure 4.12. The effect of polyamine antagonists, arcaine and ifenprodil upon

spermine’s ability to stimulate methionine synthase activity. The con cen tration o f

a rcain e w a s 6 16 fiM, the con cen tration o f ifen prodil w a s 2 0 8 p M a n d the con cen tration o f sperm in e w a s 3 pM . The rea ctio n w a s p e rfo rm e d in 5 0 m M h epes buffer p H 7.2 a t 3 7 f o r 3 0 minutes. 3 0 p g o f p ro te in w a s u sed in each assay. A ll values a re m eans ± S D ; * 0 .0 2 < P < 0 .0 5 ; ** P < 0 .0 0 1 using a tw o s a m p le d t- te st a n d co m p a rin g w ith co n tro l va lu es (0%). There w a s no sign ifican t difference b etw een sperm in e a n d sperm in e w ith a n ta g o n ists using a tw o sa m p le d t-te st n = 2 f o r a ll valu es in clu din g controls..

The results shown in figure 5.12. show that the antagonists had no inhibitory

effect upon the ability of spermine to stimulate enzyme activity; indeed a small, non

significant, enhancement in the degree of stimulation caused by spermine was observed.

This result suggests that the polyamine binding site on the enzyme is different to that at

the NMDA receptor channel.

Chapter 5.____________________________________________________________________ Polyamines.

Conclusions.

Although polyamines were previously found not to affect methionine synthase

activity when the assay was performed in phosphate buffer, they did stimulate enzyme

activity in other buffering systems. Reducing the concentration o f phosphate enabled

stimulation o f methionine synthase activity by spermine. The degree o f stimulation was

greatly reduced, however. This suggested that phosphate ions interfered with the ability

o f spermine (and the other polyamines) to stimulate enzyme activity, through some

unknown mechanism.

Vitamin B 12-dependent methionine synthase activity was reduced in hepes or tris

buffers. The length o f time for which the enzyme could be stored in these buffers was

also greatly reduced. This suggested that phosphate ions might have had a stabilising

effect upon the enzyme.

N-acetylpolyamines were also discovered to stimulate the activity o f methionine

synthase. A rank order o f potency o f the polyamines was found in which spermine >

spermidine > putrescine = cadaverine. N-acetylpolyamines did not fit into the linear

relationship found for the polyamines. Increased charge on the N-acetylpolyamines was

still associated with increased ability to stimulate methionine synthase activity.

The ECso’s o f spermine and spermidine were determined. These were found to

be different in hepes or tris buffer. The values were: spermine in hepes buffer, 8 pM; in

tris buffer 21 pM; spermidine in hepes buffer, 40 pM; and in tris buffer 100 pM. The

ratio between these two values was the same for spermine and spermidine. This

suggests that the increase in the ECso in tris buffer was due to the same effect for

spermine and spermidine. The reason for the increase in the ECso was not determined.

One possible explanation may be that as tris is an amine compound and this may

interfere with the polyamine induced stimulation o f methionine synthase. The ECso’s for

both spermine and spermidine were within the reported physiological range for growing

cells and tumour cells suggesting that this effect may occur in vivo.

The stimulation o f methionine synthase activity was found to be linear with time

and to occur at all the time periods tested.

The effect o f spermine upon the steady state kinetics o f methionine synthase was

also investigated. Spermine was found to cause uncompetitive stimulation, a reversible

form o f stimulation. The reversibility o f the stimulation is in accordance with the

hypothesis that the ability o f the polyamines to stimulate methionine synthase activity

was based upon charge; and was therefore due to electrostatic attraction. The

stimulation also only occurs after one o f the substrates binds to the enzyme.

The effect o f antagonists (arcaine and ifenprodil) for the polyamine binding site

on the NMDA receptor channel was tested. Neither compound directly affected enzyme

activity. The effect o f spermine was not antagonised by arcaine or ifenprodil, suggesting

that the NMDA receptor channel polyamine binding site is different to that on

methionine synthase.

These results suggest that an intimate relationship exists between spermine and

spermidine and methionine synthase. It has already been reported that inhibition o f

methionine synthase activity decreases the formation o f spermine and spermidine; these

results suggest that inhibition o f polyamine synthesis decreases the activity o f

methionine synthase. Thus, it may be that a positive feedback effect exits in vivo. This

effect may be o f relevance in cell growth and differentiation or tumour cell growth, and

Chapter 5.____________________________________________________________________ Polyamines.

therefore should be investigated further. However, as the concentrations o f the

polyamines are tightly regulated a positive feedback effect may not be able to operate

under normal physiological conditions. The ability o f spermine and spermidine to

stimulate methionine synthase activity may then, be a method by which polyamines

control gene expression, protein synthesis, DNA production and transmethylation

reactions. Alternatively the stimulation o f methionine synthase activity may merely

ensure that the concentrations of substrates for the production of spermine and

spermidine are maintained at adequate levels.

Sum m ary of results

• Polyamines and N-acetyl polyamines were found to stimulate methionine synthase

activity.

• The effect was measurable in hepes or tris buffer; phosphate ions reduced stimulation

o f methionine synthase by spermine.

• The stimulation was linear with time.

• A rank order o f potency was found: spermine> spermidine> putrescine = cadaverine.

• Spermine had an ECso o f 8 pM (21 pM in tris) within the physiological concentration

range in cells.

• Spermidine had an ECso o f 40 pM (100 pM in tris) within the physiological

concentration range o f dividing cells.

• Spermine increases both the Km and the Vmax o f the enzyme.

• The polyamine antagonists: arcaine and ifenprodil, at the NMDA receptor channel,

could not antagonise the effect of spermine upon methionine synthase activity.