J. Biochem. 85 397^102 (1979)
Effect of Treatment with Hemin on Rat Liver Catalase1 Michiko HAMATO and Tokuhiko HIGASHL Department of Biochemistry, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142 Received for publication, September 16, 1978
Rats were injected with a single or repeated doses of hemin intraperitoneally, and the effect on liver catalase [EC 1.11.1.6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemicaHy. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and PH^-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.
The biosynthesis of catalase, particularly of its protein moiety, has been extensively studied in the authors' laboratory for many years. However, little evidence has been obtained on the relationship between the synthesis of apocatalase and that of heme. Porphyrinogenic drugs, such as allylisopropylacetamide 1 and allylisopropylacetylcarbamide (Sedormid), markedly induce 3-aminolevulinic acid synthetase, which is the first and rate-limiting enzyme in the biosynthetic pathway for heme (1-3). Upon administration of these drugs a reduced activity of liver catalase has been 1
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, for which the authors are greatly indebted. 1 Abbreviations: A1A, allylisopropylacetamide; 3-ALA, 3-aminolevulinic acid. Vol. 8J, No. 2.J1979
397
observed (4-9). Talman and Aldrich (7), and Goldberg and Rimington (8) reported that the decrease in liver catalase produced by AIA treatment was due to a repression in the biosynthesis of catalase, and a similar mechanism was proposed for the effect of porphyrinogenic drugs other than AIA (4, 10). In Sedormid-treated rats, Kawamata et al. (11) observed a decrease not only of catalase enzyme activity but also of catalase protein determined immunochemically. From the results in further labelling experiments with simultaneously injected [14C]leucine and ['H]5-aminolevulinic acid,1 they have suggested: (i) a repression in the synthesis of apocatalase and (ii) an inhibited combination between heme and apocatalase. Suspecting the decreased synthesis of catalase in ALA-treated rats to be caused by accumulation of heme, Satyanarayana et al. (12, 13) injected rats intraperitoneally with 2 mg of hemin per lOOg
398
M. HAMATO and T. H1GASHI
of body weight and observed a 30% decrease in liver catalase activity after 6 h. To clarify the relationship of heme to apocatalase in the course of biosynthesis of the holoenzyme, a more extended study on the synthesis of catalase in hemin-treated rats is required. On the other hand, evidence indicating a regulatory mechanism by heme for the synthesis of globin in reticulocytes has been reported (14-16). Therefore, it is interesting to investigate whether apocatalase synthesis is controlled by the amount of heme.
The change in its specific radioactivity after hemin administration was traced. Reagents—Anti-catalase antiserum was prepared as described in previous papers (17, 18, 20). L-{14C(£/)]Leucine (specific radioactivity; 348 mCi permmol) and \*H(G)]8-ALA (specific radioactivity; 2.0 Ci per mmol) were purchased from The Radiochemical Centre, Amersham, England. Disodium salts of ATP and GTP, Tris, phosphocreatine and creatine kinase were obtained from Sigma Co. Most of the reagents for scintillation fluid were from Nakarai Chemicals Co.
MATERrALS AND METHODS Administration of Hemin—Rats weighing 100200 g were injected intraperitoneally with hemin dissolved in a small amount of 1 N NaOH and diluted with phosphate buffer, pH 7.0, to give a concentration of 10 or 30 mg/ml. The dose of hemin was 2 or 6 mg per 100 g of body weight (single administration). In some of the experiments 6 mg of hemin was given three times at 12-h intervals (repeated administration). Assay of Catalase—At an indicated time after hemin injection the rats was killed, and the livers were homogenized and treated with Triton X-100 for solubilization of the enzyme. The amount of catalase in the supernatant after centrifugation of the treated homogenate at 105,000 xg for 60 min was assayed by both enzymatic and immunochemical methods. The assay methods were described in the previous papers (17,18). Double Labelling of Liver Catalase In Vivo— A mixture of [14C]leucine and PHJi-ALA was injected intravenously into a rat and the labelling of catalase in peroxisomes and cytosol of the liver was studied in the manner reported previously (19). Assay of Catalase-Synthesizing Polysomes— Immunochemical precipitation of polysomes synthesizing catalase was performed according to the method of Higashi et al. (20). The immunoprecipitates were assayed at 260 nm, after dissolution in 0.1 N NaOH. In Vitro Incorporation of [l4C]Leucine and i [ H]d-ALA into Catalase—Catalase synthesis in a cell-free system was carried out as reported previously (21). The incorporations of ["C]leucine and fH]3-ALA were measured. Degradation of Liver Catalase—Rat liver catalase was labelled with injected ["Qleucine for 15 h.
RESULTS Changes in the Level of Liver Catalase after Administration of Hemin—Figure 1 shows the amount of liver catalase at 2, 4, 6, 8, 12, and 16 h after a single injection of 2 mg of hemin per 100 g body weight of rat. Changes in the level of catalase determined enzymatically (catalase enzyme) were almost parallel with those of catalase determined immunochemically (catalase antigen). Both catalase enzyme and antigen decreased immediately after hemin injection, and fell as low as 82% (enzyme) and 76% (antigen) of the normal values in 4 h. They recovered to the original levels after 6-8 h. Such a decrease was also observed on
0
2
1
6
T 1 BE A F I E I
» 5
5
5
16
111 J E C T I On ( ran )
Fig. 1. Changes in liver catalase after the injection of 2 mg of hemin. 0.2 ml of hemin solution (10 mg/ml) was injected intraperitoneally into rats per lOOg of body weight, and the amount of catalase in total liver homogenates was assayed at various times after the injection. Data are expressed in arbitrary units (17). A, Catalase enzyme determined by enzymatic assay; • , catalase antigen determined by the immunochemical method. Standard errors are shown by bars with the number of animals in parentheses.
/. Biochem.
EFFECT OF HEMIN ON LIVER CATALASE
administration of 6 mg of hemin (Fig. 2). In the latter case the decrease occurred more rapidly, the catalase enzyme being 79% and the catalase antigen 78 % of the normal values at 2 h after the hemin injection. Effect of Repeated Administration of Hemin on
0
2
1
6
T I HE A F T E R
8
10
399 Liver Catalase—Six mg of hemin was injected intraperitoneally into rats three times at 12-h intervals, and the amount of liver catalase was determined 12 h after the last injection. As shown in Fig. 3, both catalase enzyme and antigen decreased with time, being 32% and 54% of the normal values, respectively, after 36 h. Histological examinations gave no evidence of damage to the liver cells. Double Labeling of Liver Catalase in HeminTreated Rats—The in vivo incorporation of [MC]leucine and [*H]3-ALA into liver catalase was studied with rats receiving 6 mg of hemin (a single dose). Rats were treated with hemin 2, 8, or 12 h before injection of a mixture of [14C]leucine and
12
I N J E C T I O N ( noum )
Fig. 2. Changes in liver catalase after the injection of 6 mg of hemin. 0.2 ml of hemin solution (30 mg/ml) was injected intraperitoneally into rats per 100 g of body weight. See the legend to Fig. 1. A, Catalase enzyme; • , catalase antigen.
5
200 0
5
TIRE
150-
50
12
21
36
T l f l E ( HOURS )
Fig. 3. Effect of repeated administrations of hemin on liver catalase. 6 mg of hemin was given to rats every 12 h, as indicated by arrows. The amount of catalase in total liver homogenates was assayed at 12 h after the last injection of hemin. • , Catalase enzyme; • , catalase antigen. Vol. 85, No. 2, 1979
2
1
AFTER
6
8
10
12
H E K I H I N J E C T I O N l H0UR3 )
Fig. 4. Double labelling of liver catalase in rats after treatment with 6 mg of hemin. A single dose of hemin (6 mg/100 g) was given intraperitoneally to rats. After 2, 8, or 12 h a mixture of 10 /iCi of ["Qleucine and 50 /iCi of fH^-ALA in 0.2 ml was injected through a tail vein. Thirty min later, the rats were killed and the liver homogenates were fractionated by differential centrifugation. The peroxisomal fraction was treated with Triton X-100. Anti-catalase antiserum was added to the resulting extract and the cell sap fraction to isolate catalase. " C and *H in the immunoprecipitates were measured, and the specific radioactivities per unit of catalase antigen were compared with those from control rats. A, 'H radioactivities; B, ltC radioactivities; C, catalase level in liver. —•—, Peroxisomal catalase; - - O - - , supernatant catalase.
M. HAMATO and T. H1GASHI
400
PH]5-ALA. Thirty min after the isotope injection, liver peroxisomal and supernatant catalases were separated by means of anti-catalase antibody and the incorporated radioactivities were measured. The specific radioactivities per unit amount of catalase antigen were compared with those of control rats. The results are shown in Fig. 4, expressed as percent of the control. Two h after hemin injection, when the level of catalase had fallen to the minimum, the 14C specific radioactivity of catalase increased to about 150%, whereas 3 H incorporation was rather lower than the control. However, when the amount of catalase returned to the normal level 8 h after hemin injection, the catalase label was higher in both " C and *H relative to the control. The peroxisomal and the supernatant catalases showed similar patterns in labelling. In the experiment with rats receiving repeated administration of hemin and showing a reduced level of catalase, the specific radioactivities of 14C and 3 H were about 340% and 250%, respectively, in the peroxisomal and the supernatant catalases (Fig. 5). Cataluse-Synthesizing Polysomes in HeininTreated Rats—Immunoprecipitation of catalasesynthesizing polysomes was performed with liver polysomes from rats receiving repeated administrations of 6 mg of hemin. The results were compared with those from control rats. As illustrated
in Fig. 6, the population of catalase-synthesizing polysomes was slightly higher in the hemin-treated rats. Biosynthesis of Catalase in a Cell-Free System —Liver ribosomes and the cell sap were prepared separately from both intact and hemin-treated (repeated administration) rats. Various combinations of the ribosomes and the cell sap were examined in a cell-free system for the incorporation of [14C]leucine and [*H]3-ALA into catalase. The results are summarized in Table 1. Preparations originating from hemin-treated rats, particularly the ribosomes, showed a reduced activity for incorporating [14C]leucine into newly
6.0
5,0
1.0
3.0
2.0
1.0
PEROXISOHAL
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. '
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.
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.
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.
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SUFEUATANT C.
-I
278
IK :
CATALASE LEVEL
0
100 PERCENT
200 OF
10 20 AMOUNT OF RIBOSOttES (
0
216
300
400
CONTROL
Fig. 5. Labelling of liver catalase with [14C]leucine and ['HJ3-ALA after repeated administrations of hemin. Six mg of hemin was injected into rats three times at 12 h intervals, and the rats were killed 12 h after the last injection. A mixture of the two isotopes was given 30 min prior to sacrifice. For other details, see the legend to Fig. 4.
60 A 280
)
H«m
0 721
1.778
4.775
5 .835
S.E.
0 095
0.192
0.381
0 .191
Mean
0 495
1.100
Z.971
4 .931
S.E.
0.057
0.103
0.459
0 .192
Fig. 6. Catalase-synthesizing polysomes in hemintreated rats. Rats were treated with 3 x 6 mg of hemin and killed 12 h after the last injection. Ribosomes were prepared from liver homogenates of normal and hemintreated rats, and the catalase-synthesizing polysomes in these preparations were precipitated with anti-catalase in the presence of added carrier catalase (direct method). Four concentrations of ribosomes (Aw units) were tested and the immunoprecipitates were assayed at 260 nm. • , Hemin-treated rats; O, control rats. Data are the means of four experiments, and standard errors are shown by bars.
J. Biochem.
EFFECT OF HEMIN ON LIVER CATALASE TABLE I. Synthesis of catalase in a cell-free system. Four combinations of ribosomes and cell sap were incubated with ATP, GTP, an energy-generating system, ["C]leucine, and [»H],J-ALA at 37°C for 60 min. The incorporation of radioactivities into the catalase released into the medium was measured. The system consisting of ribosomes and cell sap both from intact rats was used as a standard. N, Preparations from intact rats; H, preparations from hemin-treated rats (12 h after repeated administrations). Figures are the averages of 3-5 determinations.
Ribosomes Cell sap N H N H
"C
•H
100 88 73 77
100 95 104 102
10-
t (15)
synthesized significant PH]
Effect of Treatment with Hemin on Rat Liver Catalase1 Michiko HAMATO and Tokuhiko HIGASHL Department of Biochemistry, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142 Received for publication, September 16, 1978
Rats were injected with a single or repeated doses of hemin intraperitoneally, and the effect on liver catalase [EC 1.11.1.6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemicaHy. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and PH^-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.
The biosynthesis of catalase, particularly of its protein moiety, has been extensively studied in the authors' laboratory for many years. However, little evidence has been obtained on the relationship between the synthesis of apocatalase and that of heme. Porphyrinogenic drugs, such as allylisopropylacetamide 1 and allylisopropylacetylcarbamide (Sedormid), markedly induce 3-aminolevulinic acid synthetase, which is the first and rate-limiting enzyme in the biosynthetic pathway for heme (1-3). Upon administration of these drugs a reduced activity of liver catalase has been 1
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, for which the authors are greatly indebted. 1 Abbreviations: A1A, allylisopropylacetamide; 3-ALA, 3-aminolevulinic acid. Vol. 8J, No. 2.J1979
397
observed (4-9). Talman and Aldrich (7), and Goldberg and Rimington (8) reported that the decrease in liver catalase produced by AIA treatment was due to a repression in the biosynthesis of catalase, and a similar mechanism was proposed for the effect of porphyrinogenic drugs other than AIA (4, 10). In Sedormid-treated rats, Kawamata et al. (11) observed a decrease not only of catalase enzyme activity but also of catalase protein determined immunochemically. From the results in further labelling experiments with simultaneously injected [14C]leucine and ['H]5-aminolevulinic acid,1 they have suggested: (i) a repression in the synthesis of apocatalase and (ii) an inhibited combination between heme and apocatalase. Suspecting the decreased synthesis of catalase in ALA-treated rats to be caused by accumulation of heme, Satyanarayana et al. (12, 13) injected rats intraperitoneally with 2 mg of hemin per lOOg
398
M. HAMATO and T. H1GASHI
of body weight and observed a 30% decrease in liver catalase activity after 6 h. To clarify the relationship of heme to apocatalase in the course of biosynthesis of the holoenzyme, a more extended study on the synthesis of catalase in hemin-treated rats is required. On the other hand, evidence indicating a regulatory mechanism by heme for the synthesis of globin in reticulocytes has been reported (14-16). Therefore, it is interesting to investigate whether apocatalase synthesis is controlled by the amount of heme.
The change in its specific radioactivity after hemin administration was traced. Reagents—Anti-catalase antiserum was prepared as described in previous papers (17, 18, 20). L-{14C(£/)]Leucine (specific radioactivity; 348 mCi permmol) and \*H(G)]8-ALA (specific radioactivity; 2.0 Ci per mmol) were purchased from The Radiochemical Centre, Amersham, England. Disodium salts of ATP and GTP, Tris, phosphocreatine and creatine kinase were obtained from Sigma Co. Most of the reagents for scintillation fluid were from Nakarai Chemicals Co.
MATERrALS AND METHODS Administration of Hemin—Rats weighing 100200 g were injected intraperitoneally with hemin dissolved in a small amount of 1 N NaOH and diluted with phosphate buffer, pH 7.0, to give a concentration of 10 or 30 mg/ml. The dose of hemin was 2 or 6 mg per 100 g of body weight (single administration). In some of the experiments 6 mg of hemin was given three times at 12-h intervals (repeated administration). Assay of Catalase—At an indicated time after hemin injection the rats was killed, and the livers were homogenized and treated with Triton X-100 for solubilization of the enzyme. The amount of catalase in the supernatant after centrifugation of the treated homogenate at 105,000 xg for 60 min was assayed by both enzymatic and immunochemical methods. The assay methods were described in the previous papers (17,18). Double Labelling of Liver Catalase In Vivo— A mixture of [14C]leucine and PHJi-ALA was injected intravenously into a rat and the labelling of catalase in peroxisomes and cytosol of the liver was studied in the manner reported previously (19). Assay of Catalase-Synthesizing Polysomes— Immunochemical precipitation of polysomes synthesizing catalase was performed according to the method of Higashi et al. (20). The immunoprecipitates were assayed at 260 nm, after dissolution in 0.1 N NaOH. In Vitro Incorporation of [l4C]Leucine and i [ H]d-ALA into Catalase—Catalase synthesis in a cell-free system was carried out as reported previously (21). The incorporations of ["C]leucine and fH]3-ALA were measured. Degradation of Liver Catalase—Rat liver catalase was labelled with injected ["Qleucine for 15 h.
RESULTS Changes in the Level of Liver Catalase after Administration of Hemin—Figure 1 shows the amount of liver catalase at 2, 4, 6, 8, 12, and 16 h after a single injection of 2 mg of hemin per 100 g body weight of rat. Changes in the level of catalase determined enzymatically (catalase enzyme) were almost parallel with those of catalase determined immunochemically (catalase antigen). Both catalase enzyme and antigen decreased immediately after hemin injection, and fell as low as 82% (enzyme) and 76% (antigen) of the normal values in 4 h. They recovered to the original levels after 6-8 h. Such a decrease was also observed on
0
2
1
6
T 1 BE A F I E I
» 5
5
5
16
111 J E C T I On ( ran )
Fig. 1. Changes in liver catalase after the injection of 2 mg of hemin. 0.2 ml of hemin solution (10 mg/ml) was injected intraperitoneally into rats per lOOg of body weight, and the amount of catalase in total liver homogenates was assayed at various times after the injection. Data are expressed in arbitrary units (17). A, Catalase enzyme determined by enzymatic assay; • , catalase antigen determined by the immunochemical method. Standard errors are shown by bars with the number of animals in parentheses.
/. Biochem.
EFFECT OF HEMIN ON LIVER CATALASE
administration of 6 mg of hemin (Fig. 2). In the latter case the decrease occurred more rapidly, the catalase enzyme being 79% and the catalase antigen 78 % of the normal values at 2 h after the hemin injection. Effect of Repeated Administration of Hemin on
0
2
1
6
T I HE A F T E R
8
10
399 Liver Catalase—Six mg of hemin was injected intraperitoneally into rats three times at 12-h intervals, and the amount of liver catalase was determined 12 h after the last injection. As shown in Fig. 3, both catalase enzyme and antigen decreased with time, being 32% and 54% of the normal values, respectively, after 36 h. Histological examinations gave no evidence of damage to the liver cells. Double Labeling of Liver Catalase in HeminTreated Rats—The in vivo incorporation of [MC]leucine and [*H]3-ALA into liver catalase was studied with rats receiving 6 mg of hemin (a single dose). Rats were treated with hemin 2, 8, or 12 h before injection of a mixture of [14C]leucine and
12
I N J E C T I O N ( noum )
Fig. 2. Changes in liver catalase after the injection of 6 mg of hemin. 0.2 ml of hemin solution (30 mg/ml) was injected intraperitoneally into rats per 100 g of body weight. See the legend to Fig. 1. A, Catalase enzyme; • , catalase antigen.
5
200 0
5
TIRE
150-
50
12
21
36
T l f l E ( HOURS )
Fig. 3. Effect of repeated administrations of hemin on liver catalase. 6 mg of hemin was given to rats every 12 h, as indicated by arrows. The amount of catalase in total liver homogenates was assayed at 12 h after the last injection of hemin. • , Catalase enzyme; • , catalase antigen. Vol. 85, No. 2, 1979
2
1
AFTER
6
8
10
12
H E K I H I N J E C T I O N l H0UR3 )
Fig. 4. Double labelling of liver catalase in rats after treatment with 6 mg of hemin. A single dose of hemin (6 mg/100 g) was given intraperitoneally to rats. After 2, 8, or 12 h a mixture of 10 /iCi of ["Qleucine and 50 /iCi of fH^-ALA in 0.2 ml was injected through a tail vein. Thirty min later, the rats were killed and the liver homogenates were fractionated by differential centrifugation. The peroxisomal fraction was treated with Triton X-100. Anti-catalase antiserum was added to the resulting extract and the cell sap fraction to isolate catalase. " C and *H in the immunoprecipitates were measured, and the specific radioactivities per unit of catalase antigen were compared with those from control rats. A, 'H radioactivities; B, ltC radioactivities; C, catalase level in liver. —•—, Peroxisomal catalase; - - O - - , supernatant catalase.
M. HAMATO and T. H1GASHI
400
PH]5-ALA. Thirty min after the isotope injection, liver peroxisomal and supernatant catalases were separated by means of anti-catalase antibody and the incorporated radioactivities were measured. The specific radioactivities per unit amount of catalase antigen were compared with those of control rats. The results are shown in Fig. 4, expressed as percent of the control. Two h after hemin injection, when the level of catalase had fallen to the minimum, the 14C specific radioactivity of catalase increased to about 150%, whereas 3 H incorporation was rather lower than the control. However, when the amount of catalase returned to the normal level 8 h after hemin injection, the catalase label was higher in both " C and *H relative to the control. The peroxisomal and the supernatant catalases showed similar patterns in labelling. In the experiment with rats receiving repeated administration of hemin and showing a reduced level of catalase, the specific radioactivities of 14C and 3 H were about 340% and 250%, respectively, in the peroxisomal and the supernatant catalases (Fig. 5). Cataluse-Synthesizing Polysomes in HeininTreated Rats—Immunoprecipitation of catalasesynthesizing polysomes was performed with liver polysomes from rats receiving repeated administrations of 6 mg of hemin. The results were compared with those from control rats. As illustrated
in Fig. 6, the population of catalase-synthesizing polysomes was slightly higher in the hemin-treated rats. Biosynthesis of Catalase in a Cell-Free System —Liver ribosomes and the cell sap were prepared separately from both intact and hemin-treated (repeated administration) rats. Various combinations of the ribosomes and the cell sap were examined in a cell-free system for the incorporation of [14C]leucine and [*H]3-ALA into catalase. The results are summarized in Table 1. Preparations originating from hemin-treated rats, particularly the ribosomes, showed a reduced activity for incorporating [14C]leucine into newly
6.0
5,0
1.0
3.0
2.0
1.0
PEROXISOHAL
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. '
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SUPEWATAHT C.
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.
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SUFEUATANT C.
-I
278
IK :
CATALASE LEVEL
0
100 PERCENT
200 OF
10 20 AMOUNT OF RIBOSOttES (
0
216
300
400
CONTROL
Fig. 5. Labelling of liver catalase with [14C]leucine and ['HJ3-ALA after repeated administrations of hemin. Six mg of hemin was injected into rats three times at 12 h intervals, and the rats were killed 12 h after the last injection. A mixture of the two isotopes was given 30 min prior to sacrifice. For other details, see the legend to Fig. 4.
60 A 280
)
H«m
0 721
1.778
4.775
5 .835
S.E.
0 095
0.192
0.381
0 .191
Mean
0 495
1.100
Z.971
4 .931
S.E.
0.057
0.103
0.459
0 .192
Fig. 6. Catalase-synthesizing polysomes in hemintreated rats. Rats were treated with 3 x 6 mg of hemin and killed 12 h after the last injection. Ribosomes were prepared from liver homogenates of normal and hemintreated rats, and the catalase-synthesizing polysomes in these preparations were precipitated with anti-catalase in the presence of added carrier catalase (direct method). Four concentrations of ribosomes (Aw units) were tested and the immunoprecipitates were assayed at 260 nm. • , Hemin-treated rats; O, control rats. Data are the means of four experiments, and standard errors are shown by bars.
J. Biochem.
EFFECT OF HEMIN ON LIVER CATALASE TABLE I. Synthesis of catalase in a cell-free system. Four combinations of ribosomes and cell sap were incubated with ATP, GTP, an energy-generating system, ["C]leucine, and [»H],J-ALA at 37°C for 60 min. The incorporation of radioactivities into the catalase released into the medium was measured. The system consisting of ribosomes and cell sap both from intact rats was used as a standard. N, Preparations from intact rats; H, preparations from hemin-treated rats (12 h after repeated administrations). Figures are the averages of 3-5 determinations.
Ribosomes Cell sap N H N H
"C
•H
100 88 73 77
100 95 104 102
10-
t (15)
synthesized significant PH]
