Lifn Sciencaa, Vol . 25, pp . 553-560 Printed in the II .S .A .
Parganon Prnas
EFFECT OF DIABETES ON THE INDUCTION OF ORNITHINE DECARBOXYLASE BY REFEEDIN6 S. Jaioe Rozovski, Cheryl A. Conover, and Neil B. Rudennan From the Evens Manorial Department of Clinical Research and the Department of Medicine, Boston University Medical Canter, 75 East Newton Street, Boston, Massachusetts 02118 (Racsiwd in final forte July 5, 1979) Su~aary
Refaeding of starved rats that had previously been schedulefed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours . In diabetic rats, refeading caused a s~aller increase 1n enzyme activity in liver and none at all in muscle . When insulin was administered together with food to tM diabetic rats, ornithine decarboXylase in muscle increased to lwels greater than those observed in refed controls . The activity of the enzyme in liver also increased; howwer, the increase was still less than that observed in refed control rats . The data indicate that the induction of ornithine decarboxylase in liver and Rustle following food ingestion is altered in diabetes . In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboXylase in skeletal muscle . The activity of L-ornithine decarboxylase~(EC 4.1 .1 .17) (ODC), the ratelimiting enzyme in polyamine biosynthesis, is increased in mn~enalian tissues when protein synthesis and growth are accelerated (1,2) . In accordance with this, several groups have observed a decrease in OOC activity in rat liver An during starvation and an increase in activity during refeeding (3-5) . increased activity after feeding has also been observed in skeletal muscle (6) and liver (6,7) of undernourished rats . of great interest . The effect of diabetes on ~C activity is potential) Insulin has been shown to induce the enzyme in liver (8,9~ and gut (10), and growth hornwne and glucagon, which increase hepatic ~ activity when administered in vivo, are often increased in diabetes (11,12) . In addition, many of the me~aboTic alterations that occur following food ingestion are altered in diabetes . Despite these observations, the regulation of ODC activity in this Levine et al (13,14) have reported that ODC disorder is not wail understood . activity is increased in liver and kidneyôfrdiabetic rats ; however, Sochor et
Abbreviations : ODC ~ ornithine decarboxylase (L-ornithine carboxy-lase ; EC 4.1 .1 .17) .
0024-3205/79/060553-07$02 .00/0 Copyright (c) 1979 Perganon Prese Ltd
554
Oraithiae Decarboxylaae in Diabetes
Vol . 25, No . 6, 1979
al (15) found diminished activity in these tissues as well as in brain and Feart . In neither study was the effect of nutritional status taken into account . This report deals with the effect of diabetes on ODC activity in liver and skeletal muscle of fasted and refed rats . In addition, the effect of insulin administration on the induction of ODC by food ingestion is described . Methods Adult male Sprague-Dawley rats weighing 150 to 200 grams and fed Purina Laboratory Chow were used in all experiments . They were maintained at 21°C with 12-hour cycles of light ard .dark . When starved, they were fasted for periods up to 96 hours . In preliminary studies, we observed that not all rats started to eat at the same time when presented with food after a 48-hour fast . Therefore, refeeding was studied in rats that had been schedule-fed (allowed access to food from 9 AM to 12 AM) for seven to nine days prior to starving them for 48 hours . Ih this way, a synchronized eating response, similar in nwgnitude in the control and diabetic rats, was observed . Diabetes was produced by an intravenous injection of streptozotocin (60 mg/kg of body weight) on the fourth or fifth day of schedule feeding . This dose of streptozotocin produced mild to moderate diabetes . Blood glucose levels in the diabetic rats were 200 to 250 mgt after a meal, compared to 70 and 90 mgt in the control animals . Where indicated, insulin (regular insulin, Iletin, Lilly, 0 .2 U/100 g body weight) was injected intraperitoneally exactly 15 minutes after the initiation of refeeding . Refeeding was always started between 9 AM and 9 :30 AM . All other manipulations were perfornied under pentobarbital anesthesia (5 mg/100 g IP) . Starved rats were killed between 10 AM and 12 AM and refed rats as indicated . Hindlimb tissue, consisting mainly of gastrocnemius and posterior thigh muscles, was obtained after peeling off the surrounding skin . After that, the liver was excised and a portion of the pooled blood in the abdomen was taken for glucose analysis . Muscle and liver were homogenized immediately after excision in four and three volumes respectively of ice-cold 0 .25 M sucrose (pH 7 .2) containing 1 mM 2-mercaptoethanol and 0 .3 mM disadium EDTA, with a Polytron homogenizer (Brinkman Instruments, Westbury, New York) . The homogenate could then be frozen for periods up to 10 days without loss of enzyme activity . The homogenate was centrifuged at 40,000 g for one hour at 3 ° and the supernatant used for determination of enzyme activity . ODC was assayed by measuring the evolution of '"CO z from carboxyl-labeled ornithine using a modification of the method described by Russell and Snyder (16) . Incubations were carried out in Pyrex test tubes (100 x 16 mm) equipped with rubber stoppers from which were suspended a polyethelene center well (Kontes Glass Company, Vineland, New Jersey) . The center wells contained 0 .3 ml of hyamine hydroxide as a C0 2 -trapping reagent . The incubation mixture contained 0 .5 ~unoles of pyridoxal phosphate, 2 .5 umoles of dithiothreitol, 0 .1 ml of supernatant, 0 .5 uCi ['"C] DL-ornithine (specific activity 45 .04 mCi/mmol), and 0 .5 M tris-HCI, pH 7 .2, sufficient to make a final volume of 1 ml . Tubes were incubated in a shaking water bath at 37°C for 60 minutes, after which 0 .5 ml of 2 M citric acid was injected to stop the reaction . They were then allowed to shake for an additional 30 minutes . The '"CO Z trapped in the hyamine hydroxide was counted as described b Russell and Snyder (16) . Protein was determined according to Lowry et al (17~ . Plasma glucose was determined in a glucose analyzer (Yellow Springs~Tnstruments, Yellow Springs, Ohio) .
Vol . 25, No . 6, 1979
Ornithine Decarboxylase in Diabetes
555
Results The effect of starvation on ODC activity was initially studled in rats that had been fed ad libitum and then starved . In agreement with previously reported findings j3, ,enzyme activity in liver was markedly diminished after 24 hours of starvation . A similar pattern occurred in muscle . Of note is the finding that the initial activity of ODC in the muscle of fed rats was higher than in liver and the percentage decrease in activity with starvation was greater (figuré 1) .
W
e
s~
Z Q O
24
48
72 .
88
HOURS OF STARVATION Figure
1
Ornithine decarboxylase activity in liver and skeletal muscle of fed ad libitum and starved rats . Each data point represents the mean ± o ve to seven rats .
Refeeding produced an increase in weight of 25 to 30 grams by five hours When the in both control and diabetic schedule-fed rats (data not shown) . control group was refed after 48 hours of starvation, the activity of ODC in liver increased by 140-fold after three hours and then declined somewhat (figure 2) . Peak levels mere far greater than those observed in rats fed ad libitun (figure 1) . Refeeding caused only a six-fold increase in .ODC activ~ty~ In contrast to liver, the values achieved muscle by three hours (figure 3) . were lower than in rats fed ad libitum (figure 1) and a secondary decrease in activity was not observed . iden ca findings were obtained with 55 }aM Lornithine, a concentration far higher than that present in the extract, was added to the incubation mixture . Thus, the increase in activity was not an artifact related to changes in endogenous ornithine decarboxylase concentration .
556
Vol . 25, No . 6, 1979
Ornithine Decarbozylase in Diabetes
The effect of refeeding on the incubation of ODC was considerably altered in rats that had been made diabetic five to seven days previously (figures 2 and 3) . In liver, the increase in ODC was delayed and the peak values achieved In skeletal o~uscle, in control rats after three hours were not reached . refeeding failed to cause arty increase in activity (figure 3) .
900 600 w
~~,
J
â
x
c
a
m E,c a E W o W z
Ô ~
300 ~175
160 126 100
25 0
0
I
2
3
i
4
5
HOURS AFTER REFEEDING
Figure 2 Effect of refeeding and insulin administration on liver ornithine decarboxylase activity in control and diabetic rats . All animals had previously been schedule-fed . : ~ , control + food ; ~-----n, diabetic + food ; " , diabetic + food + insulin . Each data point represents the mean + SEM of four to six rats . See text for details .
Ornithiae Decarbo~lase ia Diabetns
Vol . 25, No . 6, 1979
557
25.0 c W .E N Q Ô
a x v~ E aô w C
a
W G
~E O tG w
20.0
15 .0
10.0 Z v O a
5.0
0 HOURS AFTER REFEEDING Figure 3 Effect of refeeding and insulin administration on skeletal muscle ornithine decarboxylase activity in control and diabetic rats . ~--~, control + food ; ~-----0, diabetic + food ; " , diabetic + food + insulin . Each data point represents the mean + SEM of four to six rats . See text and legend to Figure 2 for detâils . When exogenous insulin was administered to diabetic rats during refeading, additional increments in ODC occurred in both liver and muscle . In liver, ODC activity was greater than with refeeding alone (figure 2) ; however, the very high activities seen in control rats three hours after refeeding were not achieved . In contrast, in skeletal muscle, insulin given together with food increased ODC activity to values even greater than in the refed control group (figure 3) . The mean blood lucose level three hours after insulin aàainistratlon was 79 .0 + 16 .4 mg/dl (~ + SEM), which rules against the possibility that factors producéd in response tô hypoglycdnia caused these changes . Discussion The data indicate that the induction ôf ODC activity in liver and muscle following food ingestion is altered in diabetic rats . Refeeding failed to produce a significant increase in enzyme activity in muscle of 48-hour starved
Vol . 25, No . 6, 1979
Ornithine Decarbozylase in Diabetes
559
References U .BACHRACH, in "Function of Naturall -Occurrin Pol amines", Academic Press, New York 2. A. RAINA and JANNE. J ., Med . Biol ., 53, 121-147 (1975) . 3. S . DOMSCHKE and SOLING, .IFS,-Norm . l~etab . Res ., 5, 97-101 (1973) . 4. ELORANTA, T.O . and RAINA, A .M ., oc em . ., ,179-185 (1977) . 5 . N . FAUSTO, Biochim . Bio s . Acta ~6,T33-2(1969) . 6.h P.A . McANULT ar -i1TC .Aufn , 108, 1680-1690 (1977 ) . 7 . S.J . ROZOVSKI, ROS00, P . and WIN ,~ KT~ J~utr ., 108, 1680-1690 (1978) . 8. W.B . PANKO and KENNEY, F .T ., Biochem. Biop yTi s.~es~ommun . , 43, 345-350 (1971) . 9. L.E . MALLETTE and EXTON, J .H ., Endocrinolo , 93, 640-644 (1973) . 10 . D .V . MAUDSLEY, LEIG, J . and KOBAYA~A~-P., J . Physiol . , 231, 15571561 (1976) . 11 . R . NAVALES, PILO, A. and VIGNERI, R., Diabetes, 24, 317-327 (1975) . 12 . R .H . UMGER and ORCI, L., Ph siol . Rev .,~7T8-$~6 (1976) . 13 . J.H . LEVINE, LEAMING, A.B . an , .G ., 59th Annual Meeting of the Endocrine Society, Chicago, June, 1977 . 14 . J .H . LEVINE, BUSE, M.6 ., LEAMING, A.B . and RASKIN, P., Endocrinology , 103, 1234-1238 (1978) . 15 . M. SOCHOR, BAQUER, N .Z ., HOTHERSALL, J .S ., and McLEAN, P ., Biochem. Bio his . Res. Commun ., 80, 533-539 (1978) . 16 . .H . U an ER,S.H ., Proc . Natl . Acad . Sci . USA , 6Q, 1420-1427 (1968) . 17 . O .H . LOWRY, ROSEBROU6H, N .J ., FARR, A.L . and RANDALL, R .J ., J . Biol . Chem ., 193, 265-275 (1951) . 18 . b.A .-RU3`$ELL and SNYDER, S.H ., Molec. Pharmacol ., 5, 253-262 (1969) . 19 . I .G . WOOL, STIREWALT, W.S ., KUR , ., , .B-, BAILEY, P . and OPER, D ., Rec . P_r_o_g~._ Horen . Res ., 24, i39-208 (1968) . 20 . Y . K74RTE1f;~nt~~em .~, 253-257 (1976) . 21 . S . HAYASHI, , . an NÔGUCHI, T ., Biochem . Biophys . Res . Commun . , 46, 795-780 (1972) . 1.
r.
Parganon Prnas
EFFECT OF DIABETES ON THE INDUCTION OF ORNITHINE DECARBOXYLASE BY REFEEDIN6 S. Jaioe Rozovski, Cheryl A. Conover, and Neil B. Rudennan From the Evens Manorial Department of Clinical Research and the Department of Medicine, Boston University Medical Canter, 75 East Newton Street, Boston, Massachusetts 02118 (Racsiwd in final forte July 5, 1979) Su~aary
Refaeding of starved rats that had previously been schedulefed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours . In diabetic rats, refeading caused a s~aller increase 1n enzyme activity in liver and none at all in muscle . When insulin was administered together with food to tM diabetic rats, ornithine decarboXylase in muscle increased to lwels greater than those observed in refed controls . The activity of the enzyme in liver also increased; howwer, the increase was still less than that observed in refed control rats . The data indicate that the induction of ornithine decarboxylase in liver and Rustle following food ingestion is altered in diabetes . In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboXylase in skeletal muscle . The activity of L-ornithine decarboxylase~(EC 4.1 .1 .17) (ODC), the ratelimiting enzyme in polyamine biosynthesis, is increased in mn~enalian tissues when protein synthesis and growth are accelerated (1,2) . In accordance with this, several groups have observed a decrease in OOC activity in rat liver An during starvation and an increase in activity during refeeding (3-5) . increased activity after feeding has also been observed in skeletal muscle (6) and liver (6,7) of undernourished rats . of great interest . The effect of diabetes on ~C activity is potential) Insulin has been shown to induce the enzyme in liver (8,9~ and gut (10), and growth hornwne and glucagon, which increase hepatic ~ activity when administered in vivo, are often increased in diabetes (11,12) . In addition, many of the me~aboTic alterations that occur following food ingestion are altered in diabetes . Despite these observations, the regulation of ODC activity in this Levine et al (13,14) have reported that ODC disorder is not wail understood . activity is increased in liver and kidneyôfrdiabetic rats ; however, Sochor et
Abbreviations : ODC ~ ornithine decarboxylase (L-ornithine carboxy-lase ; EC 4.1 .1 .17) .
0024-3205/79/060553-07$02 .00/0 Copyright (c) 1979 Perganon Prese Ltd
554
Oraithiae Decarboxylaae in Diabetes
Vol . 25, No . 6, 1979
al (15) found diminished activity in these tissues as well as in brain and Feart . In neither study was the effect of nutritional status taken into account . This report deals with the effect of diabetes on ODC activity in liver and skeletal muscle of fasted and refed rats . In addition, the effect of insulin administration on the induction of ODC by food ingestion is described . Methods Adult male Sprague-Dawley rats weighing 150 to 200 grams and fed Purina Laboratory Chow were used in all experiments . They were maintained at 21°C with 12-hour cycles of light ard .dark . When starved, they were fasted for periods up to 96 hours . In preliminary studies, we observed that not all rats started to eat at the same time when presented with food after a 48-hour fast . Therefore, refeeding was studied in rats that had been schedule-fed (allowed access to food from 9 AM to 12 AM) for seven to nine days prior to starving them for 48 hours . Ih this way, a synchronized eating response, similar in nwgnitude in the control and diabetic rats, was observed . Diabetes was produced by an intravenous injection of streptozotocin (60 mg/kg of body weight) on the fourth or fifth day of schedule feeding . This dose of streptozotocin produced mild to moderate diabetes . Blood glucose levels in the diabetic rats were 200 to 250 mgt after a meal, compared to 70 and 90 mgt in the control animals . Where indicated, insulin (regular insulin, Iletin, Lilly, 0 .2 U/100 g body weight) was injected intraperitoneally exactly 15 minutes after the initiation of refeeding . Refeeding was always started between 9 AM and 9 :30 AM . All other manipulations were perfornied under pentobarbital anesthesia (5 mg/100 g IP) . Starved rats were killed between 10 AM and 12 AM and refed rats as indicated . Hindlimb tissue, consisting mainly of gastrocnemius and posterior thigh muscles, was obtained after peeling off the surrounding skin . After that, the liver was excised and a portion of the pooled blood in the abdomen was taken for glucose analysis . Muscle and liver were homogenized immediately after excision in four and three volumes respectively of ice-cold 0 .25 M sucrose (pH 7 .2) containing 1 mM 2-mercaptoethanol and 0 .3 mM disadium EDTA, with a Polytron homogenizer (Brinkman Instruments, Westbury, New York) . The homogenate could then be frozen for periods up to 10 days without loss of enzyme activity . The homogenate was centrifuged at 40,000 g for one hour at 3 ° and the supernatant used for determination of enzyme activity . ODC was assayed by measuring the evolution of '"CO z from carboxyl-labeled ornithine using a modification of the method described by Russell and Snyder (16) . Incubations were carried out in Pyrex test tubes (100 x 16 mm) equipped with rubber stoppers from which were suspended a polyethelene center well (Kontes Glass Company, Vineland, New Jersey) . The center wells contained 0 .3 ml of hyamine hydroxide as a C0 2 -trapping reagent . The incubation mixture contained 0 .5 ~unoles of pyridoxal phosphate, 2 .5 umoles of dithiothreitol, 0 .1 ml of supernatant, 0 .5 uCi ['"C] DL-ornithine (specific activity 45 .04 mCi/mmol), and 0 .5 M tris-HCI, pH 7 .2, sufficient to make a final volume of 1 ml . Tubes were incubated in a shaking water bath at 37°C for 60 minutes, after which 0 .5 ml of 2 M citric acid was injected to stop the reaction . They were then allowed to shake for an additional 30 minutes . The '"CO Z trapped in the hyamine hydroxide was counted as described b Russell and Snyder (16) . Protein was determined according to Lowry et al (17~ . Plasma glucose was determined in a glucose analyzer (Yellow Springs~Tnstruments, Yellow Springs, Ohio) .
Vol . 25, No . 6, 1979
Ornithine Decarboxylase in Diabetes
555
Results The effect of starvation on ODC activity was initially studled in rats that had been fed ad libitum and then starved . In agreement with previously reported findings j3, ,enzyme activity in liver was markedly diminished after 24 hours of starvation . A similar pattern occurred in muscle . Of note is the finding that the initial activity of ODC in the muscle of fed rats was higher than in liver and the percentage decrease in activity with starvation was greater (figuré 1) .
W
e
s~
Z Q O
24
48
72 .
88
HOURS OF STARVATION Figure
1
Ornithine decarboxylase activity in liver and skeletal muscle of fed ad libitum and starved rats . Each data point represents the mean ± o ve to seven rats .
Refeeding produced an increase in weight of 25 to 30 grams by five hours When the in both control and diabetic schedule-fed rats (data not shown) . control group was refed after 48 hours of starvation, the activity of ODC in liver increased by 140-fold after three hours and then declined somewhat (figure 2) . Peak levels mere far greater than those observed in rats fed ad libitun (figure 1) . Refeeding caused only a six-fold increase in .ODC activ~ty~ In contrast to liver, the values achieved muscle by three hours (figure 3) . were lower than in rats fed ad libitum (figure 1) and a secondary decrease in activity was not observed . iden ca findings were obtained with 55 }aM Lornithine, a concentration far higher than that present in the extract, was added to the incubation mixture . Thus, the increase in activity was not an artifact related to changes in endogenous ornithine decarboxylase concentration .
556
Vol . 25, No . 6, 1979
Ornithine Decarbozylase in Diabetes
The effect of refeeding on the incubation of ODC was considerably altered in rats that had been made diabetic five to seven days previously (figures 2 and 3) . In liver, the increase in ODC was delayed and the peak values achieved In skeletal o~uscle, in control rats after three hours were not reached . refeeding failed to cause arty increase in activity (figure 3) .
900 600 w
~~,
J
â
x
c
a
m E,c a E W o W z
Ô ~
300 ~175
160 126 100
25 0
0
I
2
3
i
4
5
HOURS AFTER REFEEDING
Figure 2 Effect of refeeding and insulin administration on liver ornithine decarboxylase activity in control and diabetic rats . All animals had previously been schedule-fed . : ~ , control + food ; ~-----n, diabetic + food ; " , diabetic + food + insulin . Each data point represents the mean + SEM of four to six rats . See text for details .
Ornithiae Decarbo~lase ia Diabetns
Vol . 25, No . 6, 1979
557
25.0 c W .E N Q Ô
a x v~ E aô w C
a
W G
~E O tG w
20.0
15 .0
10.0 Z v O a
5.0
0 HOURS AFTER REFEEDING Figure 3 Effect of refeeding and insulin administration on skeletal muscle ornithine decarboxylase activity in control and diabetic rats . ~--~, control + food ; ~-----0, diabetic + food ; " , diabetic + food + insulin . Each data point represents the mean + SEM of four to six rats . See text and legend to Figure 2 for detâils . When exogenous insulin was administered to diabetic rats during refeading, additional increments in ODC occurred in both liver and muscle . In liver, ODC activity was greater than with refeeding alone (figure 2) ; however, the very high activities seen in control rats three hours after refeeding were not achieved . In contrast, in skeletal muscle, insulin given together with food increased ODC activity to values even greater than in the refed control group (figure 3) . The mean blood lucose level three hours after insulin aàainistratlon was 79 .0 + 16 .4 mg/dl (~ + SEM), which rules against the possibility that factors producéd in response tô hypoglycdnia caused these changes . Discussion The data indicate that the induction ôf ODC activity in liver and muscle following food ingestion is altered in diabetic rats . Refeeding failed to produce a significant increase in enzyme activity in muscle of 48-hour starved
Vol . 25, No . 6, 1979
Ornithine Decarbozylase in Diabetes
559
References U .BACHRACH, in "Function of Naturall -Occurrin Pol amines", Academic Press, New York 2. A. RAINA and JANNE. J ., Med . Biol ., 53, 121-147 (1975) . 3. S . DOMSCHKE and SOLING, .IFS,-Norm . l~etab . Res ., 5, 97-101 (1973) . 4. ELORANTA, T.O . and RAINA, A .M ., oc em . ., ,179-185 (1977) . 5 . N . FAUSTO, Biochim . Bio s . Acta ~6,T33-2(1969) . 6.h P.A . McANULT ar -i1TC .Aufn , 108, 1680-1690 (1977 ) . 7 . S.J . ROZOVSKI, ROS00, P . and WIN ,~ KT~ J~utr ., 108, 1680-1690 (1978) . 8. W.B . PANKO and KENNEY, F .T ., Biochem. Biop yTi s.~es~ommun . , 43, 345-350 (1971) . 9. L.E . MALLETTE and EXTON, J .H ., Endocrinolo , 93, 640-644 (1973) . 10 . D .V . MAUDSLEY, LEIG, J . and KOBAYA~A~-P., J . Physiol . , 231, 15571561 (1976) . 11 . R . NAVALES, PILO, A. and VIGNERI, R., Diabetes, 24, 317-327 (1975) . 12 . R .H . UMGER and ORCI, L., Ph siol . Rev .,~7T8-$~6 (1976) . 13 . J.H . LEVINE, LEAMING, A.B . an , .G ., 59th Annual Meeting of the Endocrine Society, Chicago, June, 1977 . 14 . J .H . LEVINE, BUSE, M.6 ., LEAMING, A.B . and RASKIN, P., Endocrinology , 103, 1234-1238 (1978) . 15 . M. SOCHOR, BAQUER, N .Z ., HOTHERSALL, J .S ., and McLEAN, P ., Biochem. Bio his . Res. Commun ., 80, 533-539 (1978) . 16 . .H . U an ER,S.H ., Proc . Natl . Acad . Sci . USA , 6Q, 1420-1427 (1968) . 17 . O .H . LOWRY, ROSEBROU6H, N .J ., FARR, A.L . and RANDALL, R .J ., J . Biol . Chem ., 193, 265-275 (1951) . 18 . b.A .-RU3`$ELL and SNYDER, S.H ., Molec. Pharmacol ., 5, 253-262 (1969) . 19 . I .G . WOOL, STIREWALT, W.S ., KUR , ., , .B-, BAILEY, P . and OPER, D ., Rec . P_r_o_g~._ Horen . Res ., 24, i39-208 (1968) . 20 . Y . K74RTE1f;~nt~~em .~, 253-257 (1976) . 21 . S . HAYASHI, , . an NÔGUCHI, T ., Biochem . Biophys . Res . Commun . , 46, 795-780 (1972) . 1.
r.
