Characterisation o f Raldh-

Table 4.1 Activity of Raldh-2 with 13 cis RAL under different conditions

13 cis RAL K , ( ^ M ) Kat (m in ‘) KaJ^m (l^M ‘ m in 0

Light conditions 8 72 9

Dark conditions 1 0 2 2 2

This revealed that Raldh-2 is more active to 13 cis RAL as a substrate under light conditions. The increased activity o f Raldh-2 with 13 cis RAL under light conditions was further investigated, when 13 cis RAL was exposed to light for 1 and then for 5 min, and the assay conducted under dark conditions at 37°C. The results from this are shown in Table 4.2.

Table 4.2 Activity o f Raldh-2 with 13 cis RAL on exposure to light

Time (m in ) (n m o l/m in /m g )

0 266

1 401

5 792

This revealed that the activity o f Raldh-2 with 13 cis RAL nearly doubled, after 1 min and quadrupled after 5 min. This shows that perhaps the conformation o f 13 cis RAL changes on exposure to light, to a substrate configuration that Raldh-2 prefers. However, the structure o f this compound has not been identified although it warrants fiirther investigation. Normally light exposure o f the cis RAL isomers leads to

generation o f the all-trans isomer, but from observing the rate and K^, these are vastly different to those measured for the all-trans isomer.

4.3.3 Purification o f CRBP-II

Cellular retinol binding protein type II (CRBP-II) is proposed as a retinoid carrier. It is specific for retinol, the active form o f vitamin A, but is also known to carry RAL isomers. The cDNA encoding for the protein was cloned into a pT71ysS plasmid (Stratagene) that allowed it to be fused to a His-tag. The protein was expressed using the same conditions as the Raldh-2 enzyme. The protein was purified using nickel affinity chromatography, which yielded a single band at 16 kDa, the estimated

molecular mass o f the protein. The buffer for the enzyme was changed to buffer C (20mM Hepes, 150mM KCl and 2mM DTT pH 8.0), that maintained the stability of the protein for long periods o f time at 4°C. The protein is not active on its own but on incubation with substrate it is proposed to increase the affinity o f the enzyme for the RAL isomers (193). It forms a CRBP-II-RAL complex which is proposed to fit into the active site o f Raldh-2 facilitating substrate channeling or direct metabolite transfer to the active site o f Raldh-2.

4.3.4 Effect of CRBP-II ratio on Raldh-2

Initial experiments were conducted to see whether the presence o f CRBP-II had any affect on the activity o f Raldh-2 9 cis RAL and CRBP-II were incubated together for 30 min, NAD ^ and enzyme were used to initiate the reaction. To find the ratio o f CRBP-II to 9 cis RAL at which the enzyme elicited the greatest réponse, the

following ratios o f CRBP-II to 9 cis RAL were used; 1:1, 2:1, 4:1, 8:1, 10:1, and 20:1 with 9 cis RAL being kept at a constant concentration o f SOpM. The specific activities o f Raldh-2 with the ratios were recorded and are shown in Table 4.3.

Table 4.3 Effect of CRBP-II concentration on Raldh-2 activity

Ratio of CRBP-II: 9 cisRAL ( u M ) Specific activity (n m o l/m in /m g )

1:1 135 2 : 1 216 4:1 155 8: 1 93 1 0 : 1 83 2 0 : 1 77

The results revealed that a 2:1 ratio o f CRBP-II to 9C RAL elicited the greatest response. Ratios above this including; 4:1, 8:1, 10:1 and 20:1 led to a decrease in the activity o f Raldh-2. It is possible that CRBP-II at higher concentrations starts to act as an inhibitor and so blocks the active site. It is also interesting to note that at the

C hapter 4 C haracterisation o f R aldh -2

higher ratios o f CRBP-II, increased concentrations o f RAL have no effect on Raldh-2 activity.

4.3.5 Effect o f CRBP-II with RAL on Raldh-2

Since the optimum ratio o f CRB? to substrate concentration was found, assays were conducted using 9C and AT RAL as substrates in the presence o f CRBP-II (Table 4.4).

Table 4.4 Effect of RAL and CRBP-II on Raldh-2 activity

RAL isomer Km(nM) ^cfl/Km(l^M'*min‘)

9 cis RAL 3 6 2

all-trans RAL 2 5 2.5

RAL + CRBP-II Km (pM) ^cfl/K„(pM‘min‘)

9 cis RAL 2 6 3

all-trans RAL 2 7 5

The results revealed that the presence o f CRBP-II had no effect on the value but, quite surprisingly, was found to give a higher kc^t with the all-trans isomer. This apparent increase in heat was very reproducible.

4.3.6 Cofactor specificity

ALDHs require a cofactor in order to carry out their reaction. In the family o f ALDHs this can either be NAD^ or NADP^. Kinetic measurements determined in the presence o f either NAD^ or NADP^ in varying concentrations revealed that the enzyme had a lower for NAD^, 50pM as opposed to 102p.M for NADP^ (Table 4.5).

Table 4.5 Effect of different cofactors on Raldh-2 activity

Cofactor Km (UM) ^,„,(min') ^c«/Km(MM'min‘)

NAD" 50 2 0 0 4

NADP" 1 2 0 62 0.5

This revealed that Raldh-2 prefers NAD^ as a cofactor, and is consistent with the findings o f the rat homologue o f Raldh-2.

4.3.7 Inhibitor studies

A number o f inhibitors o f Raldh-2 were investigated on the purified recombinant enzyme, in order to determine their mode o f action. Citral, for instance, is a known competitive inhibitor o f ALDHs. When this compound was incubated with Raldh-2 with varying substrate concentrations, it was found that remained the same while

increased with increasing concentrations o f citral (Table 4.6).

Table 4.6 Behaviour o f citral ou Raldh-2 activity

Citral (pM ) Km (nm ol/m in/m g)

0 14 3704

50 37 3704

100 51 3704

250 67 3704

These results bare the hallmarks o f competitive inhibition, indicating that, as with other ALDH, citral competes with the substrate for the active site.

Disulfiram (tetraethylthiuram disulphide) is used therapeutically in the treatment of alcoholism. Its administration before drinking o f alcoholic beverages results in unpleasant symptoms, such as blurred vision, nausea, and flushing o f the face and neck. Disulfiram apparently acts by inhibiting ALDHs, thereby leading to the build up o f acetaldehyde. Disulfiram is thought to inhibit the enzyme through modification o f the essential sulfhydryl group at the active site to form a disulphide linkage.

Raldh-2 was pre-incubated with a range o f disulfiram concentrations in order to gain a handle on how the enzyme reacts with the substrate (Table 4.7).

Table 4.7. Exteut of iuhibitiou of Raldh-2 with disulfiram

Disulfiram (pM ) (nm ol/m in/mg) % Iuhibitiou

0 3704 0

5 2508 32

10 2372 36

Chapter 4 C haracterisation o f R aldh -2