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[67] Cortisone 5β reductase from rat liver
[67]
CORTISONE 5~ REDUCTASE FROM RAT LIVER
499
phates are soluble but have not been purified. The condensation of farnesylpyrophosphate to squalene and all the subsequent reactions require the particulate fraction and TPNH. Up to now, no success has been reported on the solubilization of these particulate enzymes. This lack of soluble enzyme preparation and the difficulties of preparing and working with the water-insoluble substrates are the major difficulties in the elucidation of the details in the later steps of sterol biogenesis.
[ 67] Cortisone 58 Reductase from Rat Liver By GORDONM. TOMKINS Mammalian liver contains two series of enzymes which catalyze the irreversible reduction, by T P N H , of the 4-5 double bond of many 4-unsaturated 3-ketosteroids to form either the 5a or 5fl-reduced product. The 5fl reductases are soluble enzymes; the 5a reductases are confined to the microsomes. The assay and partial purification of the soluble 5fl enzyme which catalyzes the reduction of cortisone (17,21-dihydroxy-4-prenene-3,11,20trione) to 5fl-dihydrocortisone will be described in detail. The same assay can be applied to the other 5fl reductases and, with minor modifications, to the 5a enzymes as well.
Assay Method
Principle. 4-5-Unsaturated 3-ketosteroids have a strong absorption in the ultraviolet at 240 m~ which disappears on reduction of the double bond. This decrease in optical density at 240 m~ can therefore be used as an assay of enzyme activity. Since cortisone is readily soluble in organic solvents, the 0D24o is determined on dichloromethane extracts of the reaction mixture both before and after incubation. T P N H is generated by a G-6-P, G-6-P dehydrogenase, T P N system. Reagents 0.05 M Tris-maleate buffer, pH 6.4. T P N (0.0048 M) (Sigma 97), 4 mg./ml, of H20. Yeast G-6-P dehydrogenase (Sigma) (1000 units/ml.), 5 mg./ml. of H20. Na G-6-P (Sigma) (0.025 M) 7.6 mg./ml. MgC12 (0.05 M). Cortisone (0.0055 M), 2 mg./ml, of redistilled methanol.
500
ENZYMES OF LIPID METABOLISM
[67]
Dichloromethane, analytical grade, purified by passage through a silica gel column. Enzyme solution.
Procedure. To a small test tube in an ice bucket are added the following solutions: 0.1 ml. of TPN, 0.1 ml. of G-6-P, 0.05 ml. of G-6-P dehydrogenase, 0.05 ml. of MgC12, 0.1 ml. of Tris buffer, 0.3 ml. of glass-distilled water, 0.025 ml. of cortisone, and 0.2 ml. of enzyme. The reagents are thoroughly mixed, and a 0.4-ml. aliquot is removed and pipetted into a 15-ml. conical centrifuge tube fitted with a ground-glass joint and stopper containing 3.0 ml. of dichloromethane and about 1 ml. of water. The centrifuge tube is stoppered and shaken vigorously by hand for about 30 seconds. The reaction mixture is incubated at 37 ° for 15 minutes, after which time another 0.4-ml. aliquot is removed and extracted with dichloromethane in the same manner as the "zero-time" sample. The two centrifuge tubes are centrifuged at 2500 r.p.m, for 5 minutes, and the supernatant aqueous phase and coagulated protein are removed by suction. The dichloromethane is then read immediately in a spectrophotometer at 240 m~ against water as a blank. 1 The A 0D24o represents the amount of cortisone metabolized during the incubation. The molar extinction coefficient of cortisone in dichloromethane at 240 m~ is approximately 15,800. Purification Procedure
Step 1. Preparation of Extract. A dozen young Sprague-Dawley rats, weighing about 150 g., are killed by decapitation. Their livers are quickly removed, weighed, chilled, and homogenized in the Waring blendor with a volume of ice-cold 0.1 M sodium phosphate buffer, pH 7.4, equal to twice the weight of the livers. All subsequent operations are carried out in the cold. The homogenate is centrifuged for 15 minutes at 10,000 X g, and the supernatant layer is decanted and saved. (To prepare both the 5fl and the microsomal 5a reductases at the same time, this fraction is centrifuged for an additional 45 minutes at 100,000 X g, and the supernatant solution is used for further purification. The pellet can be washed and used as source for the 5a reductases.) Step ~. Fractionation with Ammonium Sulfate. To the supernatant layer from the first centrifugation, a sufficient volume of a saturated solution of ammonium sulfate is added to make the final ' Water, rather than dichloromethane, is used, since the optical density of the latter rises significantly during the course of reading a number of samples.
[57]
CORTISONE 59 REDUCTASE FROM RAT LIVER
501
solution 50% saturated with ammonium sulfate. The mixture is stirred for 15 to 20 minutes and centrifuged at 10,000 X g for 15 minutes. The sediment is discarded. To the supernatant layer, more ammonium sulfate is added until the solution is 70% saturated. The mixture is stirred and centrifuged as before, and the precipitate is resuspended in 25 to 30 ml. of distilled water. Any protein that does not dissolve is discarded by centrifugation, and the solution is frozen overnight. After thawing, any precipitate which formed is discarded by centrifugation, and the remaining solution is dialyzed against 4 1. of deionized water for 4 to 5 hours. This step gives a 30 to 50% yield with approximately fourfold purification over the unfractionated supernatant obtained by ultracentrifugation of the homogenate. Step 3. Adsorption with Alumina C~, Gel. The protein concentration of the ammonium sulfate fraction is determined. A volume of alumina C~ gel suspension containing, a weight of gel equal to half that value is centrifuged, and the supernatant liquid is discarded. The protein solution is added to the gel precipitate which is dispersed into the protein solution with a glass rod. The mixture is allowed to stand for 15 to 20 minutes with occasional stirring and then centrifuged at 10,000 X g for 10 minutes. The supernatant solution is assayed for enzyme activity, and, if it has declined to 10 to 20% of its original value, it is discarded. If not, it is treated with the same amount of gel again and again assayed. The gel precipitates are combined, washed with distilled water, and eluted with successive 5-ml. volumes of 0.05 M phosphate buffer, pH 7.4. The eluates are assayed separately, and those containing the highest specific activity are combined. The effectiveness of this step is variable, but on the average the yield is 50%, and the purification is three- to fourfold the ammonium sulfate fraction. This preparation can catalyze the reduction of about 0.5 micromole of cortisone per minute per milligram of protein at 37 ° . The gel eluates have been used for most of the studies done with the enzyme. It is contaminated, to a greater or lesser degree depending on the preparation, with reductases for several other 4,5-unsaturated steroids as well as with 3a-hydroxysteroid dehydrogenase. 2 To rid the preparation of the other reductases, other steps which are difficult to reproduce and produce a poor yield, such as organic solvent fractionation, may be employed. ~ In general, however, aging for several weeks in the deepfreeze causes considerable loss in the other reductases but an insignificant change in the cortisone activity.
Step 4. Chromatography
on DiethylaminoethylceUulose
G. M. Tomkins, J. Biol. Chem. 218, 437 (1956). 3 G. M. Tomkins, J. Biol. Chem. 225, 13 (1957).
(DEAE
502
ENZYMES OF LIPID METABOLISM
[67]
Cellulose). Either the gel eluate or the ammonium sulfate fraction can be chromatographed o n DEAE cellulose. The preparation chosen is dialyzed against 4 1. of 0.01 M Tris buffer, pH 7.4, containing 0.0064 M mercaptoethanol, for 3 to 4 hours. Five grams of cellulose (obtained from Eastman Chemical Co.) is suspended in water and poured into a chromatography column, 2 X 20 cm., and water is run through until it is packed uniformly. The column is washed with about 100 column volumes of a solution 1 M in NaOH and 1 M in KC1, followed by distilled water, until there is a negative test for chloride ion and the pH has returned to that of the water. Finally, it is washed with 25 to 50 column volumes of 0.01 M Tris-HCl buffer and 0.0064 M mercaptoethanol. The enzyme solution is applied, and the column is washed further with 100 column volumes of the Tris-mercaptoethanol mixture. A salt gradient is then started. The mixing flask contains 400 ml. of the Trismercaptoethanol mixture, and the reservoir contains an equal volume of Tris-mercaptoethanol with 0.2 M KC1. The flow rate is adjusted to about 1 ml./min., and 5-ml. fractions are collected. Reductase activity is located by measuring the acid-induced fluorescence of the TPN 4 produced when T P N H and cortisone are added to aliquots of the eluates and the mixture is incubated for 10 minutes. Appropriate blanks containing T P N H alone are run simultaneously. The reductase activity is usually eluted in the 30 to 40 fraction, whereas the bulk of the protein is eluted in earlier tubes. The 3a-hydroxysteroid dehydrogenase usually contaminates the reductase fractions. The column eluates are stable for several days, but attempts to concentrate them by various procedures often leads to complete loss of activity.
Properties Substrate Specificity. In fresh preparations, the most purified fractions can catalyze the reduction of several A4-3-ketosteroids. On aging for several weeks, however, even though there is no significant loss of cortisone reductase, the other reductases may decline in .activity until they are, for all practical purposes, undetectable. This fact, together with a number of other experiments 8,5 suggests that there are multiple, specifi~ soluble 5fl reduetases, as is the case with the 5a enzymes.6 Substrate Affinity. The K~ for cortisone is about 1.0 X 10-4 M. 7 ~See Vol. IV [17]. G. M. Tomkins and J. F. Curran, to be published. *J. S. McGuire and G. M. Tomkins, J. Biol. Chem. 235, 1634 (1960). TThis value is somewhat higher than that reported earlier,a since the latter was estimated directly from substrate-velocity data, whereas the present figure is derived from reciprocal plots.
[68]
ENZYMES OF STEROID HYDROXYLATION
503
Nucleotide Specificity. T P N H cannot be replaced by DPNH, either added directly or generated enzymatically, as the reducing agent. Inhibition by TPN. The reaction is strongly inhibited by TPN, which must be removed as it is formed in order to observe reductase activity by the assay described. Affinity for TPNH. As a result of the T P N inhibition, only an approximate K~ for T P N H can be given. This value is of the order of 1 X 10-4 M. Other Inhibitors. Various a,fl-unsaturated steroids, as well as the usual sulfhydryl reagents, can inhibit the cortisone reductase reaction. However, cytochrome c, atabrine, or other reagents which might prevent a flavin-catalyzed reduction are not effective inhibitors. Reversibility. The enzyme catalyzes a unidirectional reduction, and no unsaturation of 5fl-dihydrocortisone has been observed in the presence of T P N and substrate.
[ 68]
Enzymes
of Steroid H y d r o x y l a t i o n
By MIKA HAYANO and RALPH I. DORFMAN. Steroid hydroxylases are multiple in nature and named according to position of attack on the steroid nucleus or side chain (Fig. 1). C21
I
C 2O
B
~ Fie. 1
Primary C atoms are 18, 19, and 21. Enzymes effeeting monohydroxylations at these positions are 18-hydroxylase, 19-hydroxylase, and 21hydroxylase. Secondary C atoms for which hydroxylases are known are 1, 2, 6, 7, 11, 12, 15, and 16. These allow two stereospeeifie possibilities of attachment of the - - O H function, namely ~ and fl, the ~ designation indicating the position directed behind the plane of the ..... ~eule, and the B, that directed in front of the plane of the molecule.
CORTISONE 5~ REDUCTASE FROM RAT LIVER
499
phates are soluble but have not been purified. The condensation of farnesylpyrophosphate to squalene and all the subsequent reactions require the particulate fraction and TPNH. Up to now, no success has been reported on the solubilization of these particulate enzymes. This lack of soluble enzyme preparation and the difficulties of preparing and working with the water-insoluble substrates are the major difficulties in the elucidation of the details in the later steps of sterol biogenesis.
[ 67] Cortisone 58 Reductase from Rat Liver By GORDONM. TOMKINS Mammalian liver contains two series of enzymes which catalyze the irreversible reduction, by T P N H , of the 4-5 double bond of many 4-unsaturated 3-ketosteroids to form either the 5a or 5fl-reduced product. The 5fl reductases are soluble enzymes; the 5a reductases are confined to the microsomes. The assay and partial purification of the soluble 5fl enzyme which catalyzes the reduction of cortisone (17,21-dihydroxy-4-prenene-3,11,20trione) to 5fl-dihydrocortisone will be described in detail. The same assay can be applied to the other 5fl reductases and, with minor modifications, to the 5a enzymes as well.
Assay Method
Principle. 4-5-Unsaturated 3-ketosteroids have a strong absorption in the ultraviolet at 240 m~ which disappears on reduction of the double bond. This decrease in optical density at 240 m~ can therefore be used as an assay of enzyme activity. Since cortisone is readily soluble in organic solvents, the 0D24o is determined on dichloromethane extracts of the reaction mixture both before and after incubation. T P N H is generated by a G-6-P, G-6-P dehydrogenase, T P N system. Reagents 0.05 M Tris-maleate buffer, pH 6.4. T P N (0.0048 M) (Sigma 97), 4 mg./ml, of H20. Yeast G-6-P dehydrogenase (Sigma) (1000 units/ml.), 5 mg./ml. of H20. Na G-6-P (Sigma) (0.025 M) 7.6 mg./ml. MgC12 (0.05 M). Cortisone (0.0055 M), 2 mg./ml, of redistilled methanol.
500
ENZYMES OF LIPID METABOLISM
[67]
Dichloromethane, analytical grade, purified by passage through a silica gel column. Enzyme solution.
Procedure. To a small test tube in an ice bucket are added the following solutions: 0.1 ml. of TPN, 0.1 ml. of G-6-P, 0.05 ml. of G-6-P dehydrogenase, 0.05 ml. of MgC12, 0.1 ml. of Tris buffer, 0.3 ml. of glass-distilled water, 0.025 ml. of cortisone, and 0.2 ml. of enzyme. The reagents are thoroughly mixed, and a 0.4-ml. aliquot is removed and pipetted into a 15-ml. conical centrifuge tube fitted with a ground-glass joint and stopper containing 3.0 ml. of dichloromethane and about 1 ml. of water. The centrifuge tube is stoppered and shaken vigorously by hand for about 30 seconds. The reaction mixture is incubated at 37 ° for 15 minutes, after which time another 0.4-ml. aliquot is removed and extracted with dichloromethane in the same manner as the "zero-time" sample. The two centrifuge tubes are centrifuged at 2500 r.p.m, for 5 minutes, and the supernatant aqueous phase and coagulated protein are removed by suction. The dichloromethane is then read immediately in a spectrophotometer at 240 m~ against water as a blank. 1 The A 0D24o represents the amount of cortisone metabolized during the incubation. The molar extinction coefficient of cortisone in dichloromethane at 240 m~ is approximately 15,800. Purification Procedure
Step 1. Preparation of Extract. A dozen young Sprague-Dawley rats, weighing about 150 g., are killed by decapitation. Their livers are quickly removed, weighed, chilled, and homogenized in the Waring blendor with a volume of ice-cold 0.1 M sodium phosphate buffer, pH 7.4, equal to twice the weight of the livers. All subsequent operations are carried out in the cold. The homogenate is centrifuged for 15 minutes at 10,000 X g, and the supernatant layer is decanted and saved. (To prepare both the 5fl and the microsomal 5a reductases at the same time, this fraction is centrifuged for an additional 45 minutes at 100,000 X g, and the supernatant solution is used for further purification. The pellet can be washed and used as source for the 5a reductases.) Step ~. Fractionation with Ammonium Sulfate. To the supernatant layer from the first centrifugation, a sufficient volume of a saturated solution of ammonium sulfate is added to make the final ' Water, rather than dichloromethane, is used, since the optical density of the latter rises significantly during the course of reading a number of samples.
[57]
CORTISONE 59 REDUCTASE FROM RAT LIVER
501
solution 50% saturated with ammonium sulfate. The mixture is stirred for 15 to 20 minutes and centrifuged at 10,000 X g for 15 minutes. The sediment is discarded. To the supernatant layer, more ammonium sulfate is added until the solution is 70% saturated. The mixture is stirred and centrifuged as before, and the precipitate is resuspended in 25 to 30 ml. of distilled water. Any protein that does not dissolve is discarded by centrifugation, and the solution is frozen overnight. After thawing, any precipitate which formed is discarded by centrifugation, and the remaining solution is dialyzed against 4 1. of deionized water for 4 to 5 hours. This step gives a 30 to 50% yield with approximately fourfold purification over the unfractionated supernatant obtained by ultracentrifugation of the homogenate. Step 3. Adsorption with Alumina C~, Gel. The protein concentration of the ammonium sulfate fraction is determined. A volume of alumina C~ gel suspension containing, a weight of gel equal to half that value is centrifuged, and the supernatant liquid is discarded. The protein solution is added to the gel precipitate which is dispersed into the protein solution with a glass rod. The mixture is allowed to stand for 15 to 20 minutes with occasional stirring and then centrifuged at 10,000 X g for 10 minutes. The supernatant solution is assayed for enzyme activity, and, if it has declined to 10 to 20% of its original value, it is discarded. If not, it is treated with the same amount of gel again and again assayed. The gel precipitates are combined, washed with distilled water, and eluted with successive 5-ml. volumes of 0.05 M phosphate buffer, pH 7.4. The eluates are assayed separately, and those containing the highest specific activity are combined. The effectiveness of this step is variable, but on the average the yield is 50%, and the purification is three- to fourfold the ammonium sulfate fraction. This preparation can catalyze the reduction of about 0.5 micromole of cortisone per minute per milligram of protein at 37 ° . The gel eluates have been used for most of the studies done with the enzyme. It is contaminated, to a greater or lesser degree depending on the preparation, with reductases for several other 4,5-unsaturated steroids as well as with 3a-hydroxysteroid dehydrogenase. 2 To rid the preparation of the other reductases, other steps which are difficult to reproduce and produce a poor yield, such as organic solvent fractionation, may be employed. ~ In general, however, aging for several weeks in the deepfreeze causes considerable loss in the other reductases but an insignificant change in the cortisone activity.
Step 4. Chromatography
on DiethylaminoethylceUulose
G. M. Tomkins, J. Biol. Chem. 218, 437 (1956). 3 G. M. Tomkins, J. Biol. Chem. 225, 13 (1957).
(DEAE
502
ENZYMES OF LIPID METABOLISM
[67]
Cellulose). Either the gel eluate or the ammonium sulfate fraction can be chromatographed o n DEAE cellulose. The preparation chosen is dialyzed against 4 1. of 0.01 M Tris buffer, pH 7.4, containing 0.0064 M mercaptoethanol, for 3 to 4 hours. Five grams of cellulose (obtained from Eastman Chemical Co.) is suspended in water and poured into a chromatography column, 2 X 20 cm., and water is run through until it is packed uniformly. The column is washed with about 100 column volumes of a solution 1 M in NaOH and 1 M in KC1, followed by distilled water, until there is a negative test for chloride ion and the pH has returned to that of the water. Finally, it is washed with 25 to 50 column volumes of 0.01 M Tris-HCl buffer and 0.0064 M mercaptoethanol. The enzyme solution is applied, and the column is washed further with 100 column volumes of the Tris-mercaptoethanol mixture. A salt gradient is then started. The mixing flask contains 400 ml. of the Trismercaptoethanol mixture, and the reservoir contains an equal volume of Tris-mercaptoethanol with 0.2 M KC1. The flow rate is adjusted to about 1 ml./min., and 5-ml. fractions are collected. Reductase activity is located by measuring the acid-induced fluorescence of the TPN 4 produced when T P N H and cortisone are added to aliquots of the eluates and the mixture is incubated for 10 minutes. Appropriate blanks containing T P N H alone are run simultaneously. The reductase activity is usually eluted in the 30 to 40 fraction, whereas the bulk of the protein is eluted in earlier tubes. The 3a-hydroxysteroid dehydrogenase usually contaminates the reductase fractions. The column eluates are stable for several days, but attempts to concentrate them by various procedures often leads to complete loss of activity.
Properties Substrate Specificity. In fresh preparations, the most purified fractions can catalyze the reduction of several A4-3-ketosteroids. On aging for several weeks, however, even though there is no significant loss of cortisone reductase, the other reductases may decline in .activity until they are, for all practical purposes, undetectable. This fact, together with a number of other experiments 8,5 suggests that there are multiple, specifi~ soluble 5fl reduetases, as is the case with the 5a enzymes.6 Substrate Affinity. The K~ for cortisone is about 1.0 X 10-4 M. 7 ~See Vol. IV [17]. G. M. Tomkins and J. F. Curran, to be published. *J. S. McGuire and G. M. Tomkins, J. Biol. Chem. 235, 1634 (1960). TThis value is somewhat higher than that reported earlier,a since the latter was estimated directly from substrate-velocity data, whereas the present figure is derived from reciprocal plots.
[68]
ENZYMES OF STEROID HYDROXYLATION
503
Nucleotide Specificity. T P N H cannot be replaced by DPNH, either added directly or generated enzymatically, as the reducing agent. Inhibition by TPN. The reaction is strongly inhibited by TPN, which must be removed as it is formed in order to observe reductase activity by the assay described. Affinity for TPNH. As a result of the T P N inhibition, only an approximate K~ for T P N H can be given. This value is of the order of 1 X 10-4 M. Other Inhibitors. Various a,fl-unsaturated steroids, as well as the usual sulfhydryl reagents, can inhibit the cortisone reductase reaction. However, cytochrome c, atabrine, or other reagents which might prevent a flavin-catalyzed reduction are not effective inhibitors. Reversibility. The enzyme catalyzes a unidirectional reduction, and no unsaturation of 5fl-dihydrocortisone has been observed in the presence of T P N and substrate.
[ 68]
Enzymes
of Steroid H y d r o x y l a t i o n
By MIKA HAYANO and RALPH I. DORFMAN. Steroid hydroxylases are multiple in nature and named according to position of attack on the steroid nucleus or side chain (Fig. 1). C21
I
C 2O
B
~ Fie. 1
Primary C atoms are 18, 19, and 21. Enzymes effeeting monohydroxylations at these positions are 18-hydroxylase, 19-hydroxylase, and 21hydroxylase. Secondary C atoms for which hydroxylases are known are 1, 2, 6, 7, 11, 12, 15, and 16. These allow two stereospeeifie possibilities of attachment of the - - O H function, namely ~ and fl, the ~ designation indicating the position directed behind the plane of the ..... ~eule, and the B, that directed in front of the plane of the molecule.