반응 #318162

반응 방정식

atRA

13cRA

4-HPR

all-trans Retinoic acid

13-cis-Retinoic acid

N-(4-hydroxyphenyl)retinamide

Tris

atRA

13cRA

4-HPR

all-trans-retinol

Tris

DPPC

DTT

all-trans-retinol

Tris

DPPC

DTT

all-trans-retinol

→

11-cis-retinol

반응 조건

후처리

1. 1
   기타A control reaction mixture without any inhibitor
2. 2
   workup.WAITwas also incubated at room temperature for 15 min
3. 3
   workup.WAITincubated at room temperature for 30 min
4. 4
   기타At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched
5. 5
   workup.ADDITIONthe equal addition of all-trans-retinol [11,12-3H2]
6. 6
   기타After this the control reaction mixture
7. 7
   기타Now all the reaction mixtures
8. 8
   기타At the end of this incubation period the 200 μL reaction mixture
9. 9
   기타was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution
10. 10
    workup.ADDITIONwas added
11. 11
    workup.ADDITION500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids

실험 절차

Effect of all-trans Retinoic acid (atRA), 13-cis-Retinoic acid (13cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR) on IMH: To 1 mL of buffered suspension of RPE membranes (100 mM Tris pH 8.0, 76.7 μg of protein) was added 60 μM or 6 μm of atRA, 13cRA or 4-HPR and incubated at room temp. for 15 min. A control reaction mixture without any inhibitor was also incubated at room temperature for 15 min. At the end of the 15 min incubation, all-trans-retinol [11-12-3H2] (0.2 μM) was added to the reaction mixtures (100 mM Tris pH 8.0, 76.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT and 0.2 μM all-trans-retinol [11-12-3H2]) and incubated at room temperature for 30 min. At the end of this 30 minutes of incubation, an aliquot of the reactions were quenched to verify the equal addition of all-trans-retinol [11,12-3H2] and the effect of these inhibitors on LRAT. After this the control reaction mixture was incubated with atRA (60 & 6 μM), 13cRA (60 & 6 μM) or 4-HPR (60 & 6 μM) for 15 min. Now all the reaction mixtures were incubated with 30 μM of apo-rCRALBP (100 mM Tris pH 8.0, 7.7 μg of RPE protein, 0.2% BSA 100 μM of DPPC, 1 mM of DTT 30 μM apo-rCRALBP and 0.2 μM all-trans-retinol [11-12-3H2]) at 37° C. for 30 minutes. At the end of this incubation period the 200 μL reaction mixture was quenched by the addition of 750 μL ice cold methanol after which 100 μL of 1M sodium chloride solution was added, and 500 μl hexane (containing butylated hydroxy toluene at 1 mg/mL) was added to effect extraction of the retinoids. The retinoids were analyzed as previously described (27). The amount of 11-cis-retinol formed was used as a measurement of IMH activity. All experiments were performed in triplicate and the average values of these measurements were used for analysis.

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