Molecular and CellularEndocrinology, 6 (1977) 309-3 18 0 Elsevier/North-Holland Scientific Publishers, Ltd.
ANDROGEN REGULATION KIDNEY Charles D. KOCHAKIAN
OF TRANSFERASE
II ACTIVITY IN THE MOUSE
and T. MAYUMI
Laboratory of Experimental Endocrinology, Universityof Alabama in Birmingham, Birmingham, Alabama 35294, U.S.A. Received 17 May 1976; accepted 10 August 1976
Castration of adult male mice reduced the ability of the transferase factors of the kidney to stimulate amino acid incorporation by polysomes in vitro. The administration of testosterone propionate (TP) to the mice for 11 days greatly increased the activity of the transferase fraction. The changes occurred only in transferase II. The induction of the increase in transferase activity was evident 24 h after the injection of TP and required a lag period of at least 12 h. The concentration of pH 5 enzymes (protein) was slightly decreased by castration and was restored by TP administration. The radioactivity in the hot perchloric acid extract of the protein after amino acid incorporation was increased but the activity of the pH 5 enzyme fraction on amino incorporation was not significantly changed by androgen administration. Keywords: kidney; testosterone; zymes; mice.
transferase I; transferase II; protein synthesis; pH 5 en-
Castration decreases and testosterone propionate (TP) administration increases a factor(s) in the mouse kidney cytosol concerned with amino acid incorporation by polysomes (Kochakian and Hama, 1969; Kochakian et al., 1974). Since the pH 5 enzymes and transferases of the cytosol are essential factors in the amino acid incorporating system, the influence of castration and testosterone on these separate factors were studied.
MATERIALS
AND METHODS
Reagents Sucrose (special enzyme grade) was purchased from Mann Research Laboratories; Sodium deoxycholate from Nutritional Biochemical Co.; testosterone propionate (TP) from G.D. Searle Co.; [U-r4C]-Lleucine, 250 &i/120 pg, from New England Nuclear; dithiothreitol (DTT) from Calbiochem; ATP and GTP were from P-L Biochemicals and the phospocreatine and phosphocreatine kinase were from Sigma. 309
310
CD. Kochakian
Mice Two-month-old male mice purchased from Southern Animal Farms were maintained in an air-conditioned room at 26-28°C with regulated constant artificial light of 13 h/day. They were fed Purina Chow ad libitum. Castration was performed under sodium pentobarbital anesthesia. Tissue preparation The food was removed at 17 : 00 and the mice were autopsied 16 h later. The mice were killed by separation of the spinal cord at the case of the skull and immediately bled from the blood vessels of the neck. The kidneys were removed, weighed, and placed in a homogenizer tube containing 2 ml of an ice-cold medium consisting of 0.25 M sucrose, 0.0075 M magnesium acetate, 0.100 M KCl, 0.035 M Tris-HCl (pH 7.6) and 0.06 M mercaptoethanol (Medium A). Sufficient medium was added to give a 1 : 5 (W/V) ratio of kidney weight to volume of medium. The kidneys were crushed with the Teflon homogenizer pestle and homogenization was completed with 10 strokes of the pestle in 20 sec. The homogenate was centrifuged at 3000 rev./min for 20 min in the No. 860 rotor of a refrigerated International centrifuge to remove unbroken cells, nuclei and mitochondria. Preparation of polyribosomes The post-mitochondrial supernatant fraction was centrifuged at 105,OOOg for 60 min to separate the microsomes and the cytosol. The microsomes were treated with 0.5% sodium deoxycholate and the polysomes were sedimented by centrifugation at 105,OOOg for 4 h in a 0.5 M/2.0 M double sucrose gradient in Medium A (Kochakian et al, 1969). The monosomes and polysomes (ribosomes) were sedimented together by the substitution of 1.O M for the 2.0 M sucrose solution in the double gradient. The pH 5 enzyme fraction was obtained Preparation of the pH 5 enzyme fraction from the 105,OOOg supernatant fraction (cytosol). The cytosol was adjusted to pH 5.1 by the addition of 1.0 N acetic acid. After standing in ice for 30 min, the suspension was centrifuged at 10,000 g for 15 min. The precipitate was resuspended in Medium A and the precipitation was repeated to remove residual transferase activity. The second precipitate was dissolved in Medium A and frozen and stored at -2oOc. The supernatant fraction from the pH 5 Preparation of transferase factors enzyme preparation was adjusted to pH 7.0, DTT was added to a final concentration of 1 mM and then fractionated with solid ammonium sulfate (Gasior and Moldave, 1965) The protein fraction precipitating between 30 and 70% ammonium sulfate saturation (at 4O’C) was collected, dissolved in Medium A without sucrose, and passed through a 2.9 X 37 cm column of Sephadex G-25 with the same medium. The first protein peak was collected,,lyophilized, dissolved in 0.5 ml of cold water, and dialyzed against Medium A. All these steps were carried out at below 5Oc.
Androgen regulation of transferaseactivity
311
Preparation of transferase I and II fractions
The procedure is a modification of that of Felicetti and Lipmann (1968) as a result of several exploratory experiments with normal mouse kidney. The hydroxylapatite gel step was omitted and adjustments were made in the eluting solution for DEAE-cellulose ct~romatography to effect a good separation of the two transferases. The 30-70% ammonium sulfate fraction was dissolved in Buffer B containing 50 mM Tris--HCl (PH 7.4), 0.1 mM EDTA, and 2.0 mM DTT at 0.5 ml/gram fresh kidney weight, and dialyzed against several changes of Buffer B, The dialyzed sample was applied to a DEAE-cellulose column (1.5 X 25 cm). The column was washed with 60 ml of Buffer B, then the transferase II activity was eluted with a 200 ml gradient of 0 to 0.2 M KC1 in Buffer B, and the transferase I activity was eluted with 100 ml of 0.3 M KC1in Buffer B.
Protein determination
The protein content of the pH 5 enzyme and transferase fractions was determined by the method of Lowry et al. (1951).
Preparation of [14C]aminoacyl-t-RNA
[U-r4C]-Lleucyl-tRNA was prepared by incubation of normal mouse liver pH 5 enzymes with ATP and [U-‘4C]-Lleucine (Moldave, 1963). The specific activity of the [U-i4C]leucyl-tRNA was approximately 3 X lo5 dpm/mg RNA. The RNA content was determined by absorption at 260 nm (DeDeken-Grenson and DeDeken, 1959).
~n~batio~:
/ U-14~]-L-~e~~ine i~co~oration This procedure was essent~~y as previously described (Kochakian et al., 1974) with substitution of pH 5 enzymes and transferases for cytosol where indicated. [U-r4C]-L-Ieucine incorporation from [U-14C]-L-Ieucyl-tRNA was the same as above except for the substitution of ~U-14C]-L-Ieucyl-t~A for [U-14~]-L-leucine and the omission of the pH 5 enzymes. Separation of protein and the radioacticity determinations were as previously described (Kochakian et al., 1974).
RESULTS Effect of ~~trutio~ and TP on the protein concentrator ase fractions
of the pH 5 and transfer-
Castration resulted in a decrease in the concentration of the proteins of both the crude pH 5 enzymes and the transferase fraction obtained from the pH 5.1 soluble fraction by precipitation between 30 and 70% (NH&SO4 saturation (fig. 1). TP administration restored the values to normal. The concentration of polysomes was even more remarkably decreased after castration and increased on TP ad~nistration to above normal in agreement with our earlier reports (Kochakian et al., 1969,1974).
C.D. Kochakian
312
12
6
p=.o5
m/g
+
4
T
64.
t-
4
2
0
0I
A
;I I C I,
P” 5
IANS. PC
C 01
s
Fig. 1. The effect of castration (c) and testosterone propionate (TP) on the concentration of the pH 5 fraction, crude transferases and polysomes of the male mouse kidney. Tire mice were castrated at 2 months of age, two weeks later a TP pellet (15 + 1 mg) was implanted subcutaneously (Kochakian, 1944) and autopsy was after 11 days. The various fractions were prepared as described in Materials and Methods. The values are the means of 3 normal (N), 7 castrated (C) and 7 TP-treated experiments which contained 3-10 N, 21-34 C and 8-16 TP-treated mice per experiment.
Contribution of the transferme fraction to protein biosynthesis Amino acid incorporation was enhanced by TP when the 3 factors (polysomes, pH 5 enzymes, transferase fractions) from the kidney were supplied to the biosynthesis system (fig. 2). The substitution of the pH 5 factors from the kidney of the castrated mice produced a small questionable decrease in the echoing effect of the androgen. The substitution of the castrate polysomes, however, was without further detectable effect. The substitution of only the transferase fraction from the castrated mice produced a sharp decrease to the castrated level. The substitution of both polysomes and transferase from the castrated mice did not produce a further significant change. Almost identical results were obtained in two other experiments where the concentration of (a) transferase was 3.0 mg and pH 5 enzymes was 1.5 mg and (b) transferase was 1.5 mg and pH 5 enzymes was 3.0 mg, The rate of amino acid incorporation was lower but parallel at the Iower concentration of the transferase fraction.
Androgen regulation of transferaseactivity
400
313
t Ave.
Trans I 1.5 mg. PHI : 1.5 mg.
i E ,” 300i w L TP ~2oo-Tr: PO: TP @-IT: TP
TP TP C
TP C C
C
C
TPP
TCP
z
Fig. 2. The influence of TP on the activities of the transferases (Tr), pH 5 enzymes (pH 5) and polysomes (PO) on the incorporation of amino acids into protein. The mice and the various factions were prepared as indicated in fig. 1. The incubation system contained 100 pg (RNA equivalents) polysomes, 1.0 I.rmol ATP, 0.2 pmol GTP, 20 rcmol phosphocreatine, 40 c(g phosphocreatine kinase, 0.05 pmol of each of the amino acids, 1 rCi (0.05 rmol) [U-%]-L-leucine, 1.5 mg pH 5 enzymes and 1.5 mg transferases. All of the components were dissolved in water. The final volume was made to 1.0 ml with Medium A. The incubation was for 30 min at 37°C The mean of Appropriate controls were included with each series of determinations. X -X the two series of experiments (A and B). Similar results were obtained in two other series of experiments at 1.5 mg transferase, 3.0 mg pH 5 enzymes and 3.0 mg transferase, 1.5 mg pH 5 enzymes.
5oo
200
Ao.
Tr :TP PO :TP pH5:TP
C TP TP
C C TP
TP TP C
TP C C
C C C
Fig. 3. The influence of TP on the activity of the pH 5 fraction of the mouse kidney. The hot perchloric acid-soluble fraction from the proteins of f%. 2 were assayed for radioactivity.
314
C.D. Kochakian
Injluence of pH 5 fraction The perchloric acid-precipitable material of the incubation mixture contains not only proteins but also several other substances including the nucleic acids. Treatment of the precipitate with hot perchloric acid hydrolyzes the nuclei acids to acidsoluble material. The RNA should include newly formed aminoacyl-tRNA (Hoagland et al., 1958) therefore, the radioactivity of this fraction was determined to obtain an indication of the rate of formation of [U-r4C]-L-leucyl-tRNA by the preparations from the castrated and treated mice. The system containing the three fac-
0.3M KCI -+i
b 0.1-0.2MtiPtdlb
f
IO
30
FLIONS
40
50
60
(5ml)
Fig. 4. Separation of transferase I from transferase II by DEAE-cellulose chromatography. The transferase fraction was prepared and submitted to chromatography as indicated under Materials and Methods. The pooled kidneys of the castrated mice weighed 13.32 g (350 mg/mouse) and the TP-treated (11 days) mice 11.70 g (777 mg/mouse). The protein concentration is indicated as absorbance at 280 nm. An aliquot (0.1 ml) from each fraction (5 ml) was assayed for amino acid incorporation in the following incubation system: 300 c(g ribosomes, 42 pg (17,700 dpm) [U-14C]-L-leucyl-tRNA, 0.2 pmol GTP, 0.1 ml transferase I or II (0.1 ml of the most active chromatographic fractions of transferase II from the previous experiment were used in the transferase I assays and of transferase I in the transferase II assays) and 0.1 ml of the respective fractions. The final volume was made to 1.0 ml by the addition of medium A containing sufficient DTT to give a concentration of 0.16 mM. Incubation was for 30 min at 37°C. Similar results were obtained in 2 other experiments (cf. table 1).
Androgen regulation of transferaseactivity
315
tors from the treated mice exhibited much greater ra~oacti~ty in the hydrolyzate than that with the factors from the castrated mice (fig. 3). The substitution of the pH 5 fraction from the castrated mice reduced the radioactivity in the hydrolyzite to that of the system containing all of the factors from the castrated mice. The cross substitution of the transferases or the polysomes were without influence. SimiIar results were obtained in the two other series of experiments (see above). The pH 5 fraction was the rate-limiting factor. Separation of transferas~ I from transferase I.
DEAE-cellulose chromatography of the transferase fraction effectively separated transferase I from transferase II (fig. 4). The active fractions were pooled and aliquots were assayed to obtain the respective total activities. Transferase I from the kidneys of the castrated mice was as effective as that from the TP-treated mice in the incorporation of [U-‘4C]-Lleucine from [U-‘4C]-L-leucyl-tRNA into perchloric acid precipitable protein (table 1). Transferase II activity, however, was significantly increased by the androgen.
Induction of transferase activity by TP
Leucine incorporation into perchloric acid-precipitable material by the ribosomes was not enhanced until sometime between 12 and 24 h after the injection of the mice with TP (fig. 5). A further increase was obtained after 48 h. and the slope of the curve suggested a progresssive increase beginning shortly after 12 h. The substitution of the total cytosol gave a parallel results. As expected the cytosol from the castrated mice gave an apparent small but not significant increase. When the ribosomes from the castrated mice were substituted for those of the treated mice, the transferase factors from the treated mice were not.able to induce a significant increase in the transfer of amino acids to proteins. This was unexpected.
Table 1 Effect of TP on the ability of transferase I and transferase II to stimulate amino acid incorporation by ribosomes of mouse kidney. AIiquots of 0.1 ml of the active fractions from the chromatographic separation of transferase I from transferase II were pooled and a 0.1 ml aliquot of the pools was assayed for the respective activities as described in fig. 4 to give the average total activities. The values are the mean 2 S.E.M. for 3 experiments. The protein in the 0.1 ml aIiquots of the pooled samples for the 3 experiments were: transferase I: Castrate, 50,76;60; TP, 72,89,71 pg. Transferase II: Castrate, 326,381,4OS;TP, 324,367,292 pg. Transferase I Transferase II (dpm/mg protein) Castrate TP Diff. (%) P
16,150 + 1,710 17,640 f 1,250 l-9 NS
3410 f 142 5210 f 650 +62
ANDROGEN REGULATION KIDNEY Charles D. KOCHAKIAN
OF TRANSFERASE
II ACTIVITY IN THE MOUSE
and T. MAYUMI
Laboratory of Experimental Endocrinology, Universityof Alabama in Birmingham, Birmingham, Alabama 35294, U.S.A. Received 17 May 1976; accepted 10 August 1976
Castration of adult male mice reduced the ability of the transferase factors of the kidney to stimulate amino acid incorporation by polysomes in vitro. The administration of testosterone propionate (TP) to the mice for 11 days greatly increased the activity of the transferase fraction. The changes occurred only in transferase II. The induction of the increase in transferase activity was evident 24 h after the injection of TP and required a lag period of at least 12 h. The concentration of pH 5 enzymes (protein) was slightly decreased by castration and was restored by TP administration. The radioactivity in the hot perchloric acid extract of the protein after amino acid incorporation was increased but the activity of the pH 5 enzyme fraction on amino incorporation was not significantly changed by androgen administration. Keywords: kidney; testosterone; zymes; mice.
transferase I; transferase II; protein synthesis; pH 5 en-
Castration decreases and testosterone propionate (TP) administration increases a factor(s) in the mouse kidney cytosol concerned with amino acid incorporation by polysomes (Kochakian and Hama, 1969; Kochakian et al., 1974). Since the pH 5 enzymes and transferases of the cytosol are essential factors in the amino acid incorporating system, the influence of castration and testosterone on these separate factors were studied.
MATERIALS
AND METHODS
Reagents Sucrose (special enzyme grade) was purchased from Mann Research Laboratories; Sodium deoxycholate from Nutritional Biochemical Co.; testosterone propionate (TP) from G.D. Searle Co.; [U-r4C]-Lleucine, 250 &i/120 pg, from New England Nuclear; dithiothreitol (DTT) from Calbiochem; ATP and GTP were from P-L Biochemicals and the phospocreatine and phosphocreatine kinase were from Sigma. 309
310
CD. Kochakian
Mice Two-month-old male mice purchased from Southern Animal Farms were maintained in an air-conditioned room at 26-28°C with regulated constant artificial light of 13 h/day. They were fed Purina Chow ad libitum. Castration was performed under sodium pentobarbital anesthesia. Tissue preparation The food was removed at 17 : 00 and the mice were autopsied 16 h later. The mice were killed by separation of the spinal cord at the case of the skull and immediately bled from the blood vessels of the neck. The kidneys were removed, weighed, and placed in a homogenizer tube containing 2 ml of an ice-cold medium consisting of 0.25 M sucrose, 0.0075 M magnesium acetate, 0.100 M KCl, 0.035 M Tris-HCl (pH 7.6) and 0.06 M mercaptoethanol (Medium A). Sufficient medium was added to give a 1 : 5 (W/V) ratio of kidney weight to volume of medium. The kidneys were crushed with the Teflon homogenizer pestle and homogenization was completed with 10 strokes of the pestle in 20 sec. The homogenate was centrifuged at 3000 rev./min for 20 min in the No. 860 rotor of a refrigerated International centrifuge to remove unbroken cells, nuclei and mitochondria. Preparation of polyribosomes The post-mitochondrial supernatant fraction was centrifuged at 105,OOOg for 60 min to separate the microsomes and the cytosol. The microsomes were treated with 0.5% sodium deoxycholate and the polysomes were sedimented by centrifugation at 105,OOOg for 4 h in a 0.5 M/2.0 M double sucrose gradient in Medium A (Kochakian et al, 1969). The monosomes and polysomes (ribosomes) were sedimented together by the substitution of 1.O M for the 2.0 M sucrose solution in the double gradient. The pH 5 enzyme fraction was obtained Preparation of the pH 5 enzyme fraction from the 105,OOOg supernatant fraction (cytosol). The cytosol was adjusted to pH 5.1 by the addition of 1.0 N acetic acid. After standing in ice for 30 min, the suspension was centrifuged at 10,000 g for 15 min. The precipitate was resuspended in Medium A and the precipitation was repeated to remove residual transferase activity. The second precipitate was dissolved in Medium A and frozen and stored at -2oOc. The supernatant fraction from the pH 5 Preparation of transferase factors enzyme preparation was adjusted to pH 7.0, DTT was added to a final concentration of 1 mM and then fractionated with solid ammonium sulfate (Gasior and Moldave, 1965) The protein fraction precipitating between 30 and 70% ammonium sulfate saturation (at 4O’C) was collected, dissolved in Medium A without sucrose, and passed through a 2.9 X 37 cm column of Sephadex G-25 with the same medium. The first protein peak was collected,,lyophilized, dissolved in 0.5 ml of cold water, and dialyzed against Medium A. All these steps were carried out at below 5Oc.
Androgen regulation of transferaseactivity
311
Preparation of transferase I and II fractions
The procedure is a modification of that of Felicetti and Lipmann (1968) as a result of several exploratory experiments with normal mouse kidney. The hydroxylapatite gel step was omitted and adjustments were made in the eluting solution for DEAE-cellulose ct~romatography to effect a good separation of the two transferases. The 30-70% ammonium sulfate fraction was dissolved in Buffer B containing 50 mM Tris--HCl (PH 7.4), 0.1 mM EDTA, and 2.0 mM DTT at 0.5 ml/gram fresh kidney weight, and dialyzed against several changes of Buffer B, The dialyzed sample was applied to a DEAE-cellulose column (1.5 X 25 cm). The column was washed with 60 ml of Buffer B, then the transferase II activity was eluted with a 200 ml gradient of 0 to 0.2 M KC1 in Buffer B, and the transferase I activity was eluted with 100 ml of 0.3 M KC1in Buffer B.
Protein determination
The protein content of the pH 5 enzyme and transferase fractions was determined by the method of Lowry et al. (1951).
Preparation of [14C]aminoacyl-t-RNA
[U-r4C]-Lleucyl-tRNA was prepared by incubation of normal mouse liver pH 5 enzymes with ATP and [U-‘4C]-Lleucine (Moldave, 1963). The specific activity of the [U-i4C]leucyl-tRNA was approximately 3 X lo5 dpm/mg RNA. The RNA content was determined by absorption at 260 nm (DeDeken-Grenson and DeDeken, 1959).
~n~batio~:
/ U-14~]-L-~e~~ine i~co~oration This procedure was essent~~y as previously described (Kochakian et al., 1974) with substitution of pH 5 enzymes and transferases for cytosol where indicated. [U-r4C]-L-Ieucine incorporation from [U-14C]-L-Ieucyl-tRNA was the same as above except for the substitution of ~U-14C]-L-Ieucyl-t~A for [U-14~]-L-leucine and the omission of the pH 5 enzymes. Separation of protein and the radioacticity determinations were as previously described (Kochakian et al., 1974).
RESULTS Effect of ~~trutio~ and TP on the protein concentrator ase fractions
of the pH 5 and transfer-
Castration resulted in a decrease in the concentration of the proteins of both the crude pH 5 enzymes and the transferase fraction obtained from the pH 5.1 soluble fraction by precipitation between 30 and 70% (NH&SO4 saturation (fig. 1). TP administration restored the values to normal. The concentration of polysomes was even more remarkably decreased after castration and increased on TP ad~nistration to above normal in agreement with our earlier reports (Kochakian et al., 1969,1974).
C.D. Kochakian
312
12
6
p=.o5
m/g
+
4
T
64.
t-
4
2
0
0I
A
;I I C I,
P” 5
IANS. PC
C 01
s
Fig. 1. The effect of castration (c) and testosterone propionate (TP) on the concentration of the pH 5 fraction, crude transferases and polysomes of the male mouse kidney. Tire mice were castrated at 2 months of age, two weeks later a TP pellet (15 + 1 mg) was implanted subcutaneously (Kochakian, 1944) and autopsy was after 11 days. The various fractions were prepared as described in Materials and Methods. The values are the means of 3 normal (N), 7 castrated (C) and 7 TP-treated experiments which contained 3-10 N, 21-34 C and 8-16 TP-treated mice per experiment.
Contribution of the transferme fraction to protein biosynthesis Amino acid incorporation was enhanced by TP when the 3 factors (polysomes, pH 5 enzymes, transferase fractions) from the kidney were supplied to the biosynthesis system (fig. 2). The substitution of the pH 5 factors from the kidney of the castrated mice produced a small questionable decrease in the echoing effect of the androgen. The substitution of the castrate polysomes, however, was without further detectable effect. The substitution of only the transferase fraction from the castrated mice produced a sharp decrease to the castrated level. The substitution of both polysomes and transferase from the castrated mice did not produce a further significant change. Almost identical results were obtained in two other experiments where the concentration of (a) transferase was 3.0 mg and pH 5 enzymes was 1.5 mg and (b) transferase was 1.5 mg and pH 5 enzymes was 3.0 mg, The rate of amino acid incorporation was lower but parallel at the Iower concentration of the transferase fraction.
Androgen regulation of transferaseactivity
400
313
t Ave.
Trans I 1.5 mg. PHI : 1.5 mg.
i E ,” 300i w L TP ~2oo-Tr: PO: TP @-IT: TP
TP TP C
TP C C
C
C
TPP
TCP
z
Fig. 2. The influence of TP on the activities of the transferases (Tr), pH 5 enzymes (pH 5) and polysomes (PO) on the incorporation of amino acids into protein. The mice and the various factions were prepared as indicated in fig. 1. The incubation system contained 100 pg (RNA equivalents) polysomes, 1.0 I.rmol ATP, 0.2 pmol GTP, 20 rcmol phosphocreatine, 40 c(g phosphocreatine kinase, 0.05 pmol of each of the amino acids, 1 rCi (0.05 rmol) [U-%]-L-leucine, 1.5 mg pH 5 enzymes and 1.5 mg transferases. All of the components were dissolved in water. The final volume was made to 1.0 ml with Medium A. The incubation was for 30 min at 37°C The mean of Appropriate controls were included with each series of determinations. X -X the two series of experiments (A and B). Similar results were obtained in two other series of experiments at 1.5 mg transferase, 3.0 mg pH 5 enzymes and 3.0 mg transferase, 1.5 mg pH 5 enzymes.
5oo
200
Ao.
Tr :TP PO :TP pH5:TP
C TP TP
C C TP
TP TP C
TP C C
C C C
Fig. 3. The influence of TP on the activity of the pH 5 fraction of the mouse kidney. The hot perchloric acid-soluble fraction from the proteins of f%. 2 were assayed for radioactivity.
314
C.D. Kochakian
Injluence of pH 5 fraction The perchloric acid-precipitable material of the incubation mixture contains not only proteins but also several other substances including the nucleic acids. Treatment of the precipitate with hot perchloric acid hydrolyzes the nuclei acids to acidsoluble material. The RNA should include newly formed aminoacyl-tRNA (Hoagland et al., 1958) therefore, the radioactivity of this fraction was determined to obtain an indication of the rate of formation of [U-r4C]-L-leucyl-tRNA by the preparations from the castrated and treated mice. The system containing the three fac-
0.3M KCI -+i
b 0.1-0.2MtiPtdlb
f
IO
30
FLIONS
40
50
60
(5ml)
Fig. 4. Separation of transferase I from transferase II by DEAE-cellulose chromatography. The transferase fraction was prepared and submitted to chromatography as indicated under Materials and Methods. The pooled kidneys of the castrated mice weighed 13.32 g (350 mg/mouse) and the TP-treated (11 days) mice 11.70 g (777 mg/mouse). The protein concentration is indicated as absorbance at 280 nm. An aliquot (0.1 ml) from each fraction (5 ml) was assayed for amino acid incorporation in the following incubation system: 300 c(g ribosomes, 42 pg (17,700 dpm) [U-14C]-L-leucyl-tRNA, 0.2 pmol GTP, 0.1 ml transferase I or II (0.1 ml of the most active chromatographic fractions of transferase II from the previous experiment were used in the transferase I assays and of transferase I in the transferase II assays) and 0.1 ml of the respective fractions. The final volume was made to 1.0 ml by the addition of medium A containing sufficient DTT to give a concentration of 0.16 mM. Incubation was for 30 min at 37°C. Similar results were obtained in 2 other experiments (cf. table 1).
Androgen regulation of transferaseactivity
315
tors from the treated mice exhibited much greater ra~oacti~ty in the hydrolyzate than that with the factors from the castrated mice (fig. 3). The substitution of the pH 5 fraction from the castrated mice reduced the radioactivity in the hydrolyzite to that of the system containing all of the factors from the castrated mice. The cross substitution of the transferases or the polysomes were without influence. SimiIar results were obtained in the two other series of experiments (see above). The pH 5 fraction was the rate-limiting factor. Separation of transferas~ I from transferase I.
DEAE-cellulose chromatography of the transferase fraction effectively separated transferase I from transferase II (fig. 4). The active fractions were pooled and aliquots were assayed to obtain the respective total activities. Transferase I from the kidneys of the castrated mice was as effective as that from the TP-treated mice in the incorporation of [U-‘4C]-Lleucine from [U-‘4C]-L-leucyl-tRNA into perchloric acid precipitable protein (table 1). Transferase II activity, however, was significantly increased by the androgen.
Induction of transferase activity by TP
Leucine incorporation into perchloric acid-precipitable material by the ribosomes was not enhanced until sometime between 12 and 24 h after the injection of the mice with TP (fig. 5). A further increase was obtained after 48 h. and the slope of the curve suggested a progresssive increase beginning shortly after 12 h. The substitution of the total cytosol gave a parallel results. As expected the cytosol from the castrated mice gave an apparent small but not significant increase. When the ribosomes from the castrated mice were substituted for those of the treated mice, the transferase factors from the treated mice were not.able to induce a significant increase in the transfer of amino acids to proteins. This was unexpected.
Table 1 Effect of TP on the ability of transferase I and transferase II to stimulate amino acid incorporation by ribosomes of mouse kidney. AIiquots of 0.1 ml of the active fractions from the chromatographic separation of transferase I from transferase II were pooled and a 0.1 ml aliquot of the pools was assayed for the respective activities as described in fig. 4 to give the average total activities. The values are the mean 2 S.E.M. for 3 experiments. The protein in the 0.1 ml aIiquots of the pooled samples for the 3 experiments were: transferase I: Castrate, 50,76;60; TP, 72,89,71 pg. Transferase II: Castrate, 326,381,4OS;TP, 324,367,292 pg. Transferase I Transferase II (dpm/mg protein) Castrate TP Diff. (%) P
16,150 + 1,710 17,640 f 1,250 l-9 NS
3410 f 142 5210 f 650 +62
