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Coordinate behavior of orotate phosphoribosyltransferase and orotidylate decarboxylase in developing mouse liver and brain
Pergamon Prese
Lifn Sciences, Vol . 22, pp . 577-582 Printed in the U .S .A .
COORDINATE BEHAVIOR OF OROTATli PHOSPHORIHOSYLTRANSFERASE AND OROTIDYLATE DECARBOXYLASE IN DEVELOPING MOUSE LIVER AND BRAIN Philip Reyesl and Clare Intress Department of Biochemistry The University of New Mexico School of Medicine Albuquerque, New Mexico 87131 (Rrcaived in final form December 19, 1977) SUMMARY A large body of data has accumulated in recent years supporting the view that orotate phosphoribosyltransferase and orotidylate decarboxylase exist as a bif~ctional enzyme complex in adult mamma lian tissues . This paper presents evidence that such a complex also occurs in mouse liver and brain, regardless of the developmental stage of the animal . Orotate phosphoribosyltransferase and orotidylate decarboxylase activities remained coordinate in fetal, neonatal, immature and adult liver and brain . In addition, these two enzymes routinely cosedimented during centrifugation of cell-free extracts in sucrose gradients . The sedimentation coefficient of the enzyme complex did not change significantly during mouse development . However, the liver complex exhibited a sedimentation coefficient (5 .0 t 0 .2) that differed from that of the brain complex (4 .3 t 0 .1 .) INTRODUCTION It is now well established that the de novo synthesis of UMP involves the sequential action of orotate PRTase2 (EC 2 .4 .2 .10) (reaction 1) and OMP decarboxylase (EC 4 .1 .1 .23) (reaction 2 .) 2+ orotate + PP-ribose-P ,~- OMP + PPi (1) OMP
UMP + C02
(2)
An interesting feature of these two enzymes is their capacity to copurify from all mammalian tissues so far examined . These tissues include bovine brain (1), erythrocytes (2) and thymus (3), Ehrlich ascites carcinoma (4), marine leukemia P1534J (5) and human erythrocytes (6) . In addition, the activities of the above enzymes show a coordinate relationship in rat (7-9) and human (10) tissues and in erythrocytes from various mammalian species (11) . It is also well established that administration to rats and humans of the xanthine oxidase inhibitor, allopurinol (4-hydroxypyrazolo (3,4-d) pyrimidine), produces a coordinate increase in the activities of both enzymes (9,10,12) . 1. 2.
To whom reprint requests should be addressed . Abbreviations : PRTase, phosphoribosyltransferase ; OMP, orotidine 5'-monophosphate ; PP-ribose-P, 5-phosphoribosyl 1-pyrophosphate . 577
578
Coordinate Behavior of Pyrimidine Enzymaa
Vol . 22, No . 7, 1978
In view of the above findings, it is now generally believed that orotate PRTase and OMP decarboxylase exist as a bifunctional enzyme complex in mammals . It is to be noted, however, that all previous studies of this complex have emphasized the use of adult tissues . It was therefore of interest to examine fetal, neonatal, immature as well as adult mouse liver and brain in order to gather data on the occurrence of this enzyme complex during growth and development . An equally important goal of this study was to quantitate the level of activity of these two enzymes in developing mouse liver and brain . MATERIALS AND METHODS Animals . CD2Fl hybrid mice were used throughout this study . These mice representtree offspring of BALB/c inbred females mated with DBA/2 inbred males . Pregnant BALB/c mice, obtained from Lab Supply Co ., Indianapolis, Indiana, were caged individually, fed ad libitum , and housed in a room illuminated from 9 p .m . to 9 a .m . Gestationalâge was estimated retrospectively based on the day of birth of the remaining litters, all of which were born within a period of 2 days . The error in gestational age is therefore about t 1 day . Mice were weaned at 20 days of age and were segregated according to sex (6 mice per cage) 35 days after birth . Experimental data points on day -8, -1, 1, 3, 10, 21 and 35 were each obtained with pooled brains and livers from an entire litter (average of 9 animals per litter) whereas 3 males and 3 females (not necessarily from the same litter) were used as the source of pooled tissues on days 63 and 90 . Preparation of Cell-free Extracts . All animals were routinely sacrificed by decapitation during the last 30 min of the light period . Brains and livers were excised quickly, rinsed with ice-cold 0 .15 M NaCl, gently bottled, weighed and minced . In the case of pregnant mice, the uterus was removed in toto following which the fetuses were immediately delivered . All subsequent steps were done at 0-5 ° . The minced tissues were then suspended in 5 volumes of 50 mM potassium phosphate (pH 8 .0) containing 1 mM'PP-ribose-P and 1 mM dithiothreitol . Cells were broken with 7 strokes of a glass-Teflon homogenizer . The resulting suspension was centrifuged at 105,000 x g for 60 min and the supernatant fraction removed with as little of the overlying lipid as possible . This fraction, which served as the source of orotate PRTase and OMP decarboxylase, was normally assayed for enzyme activity following overnight storage at -20 ° . Analysis of this fraction by sucrose gradient centrifugation was usually done within a week of its preparation . Protein concentrations were determined by the method of Lowry et al . (13), with bovine serum albumin as standard . Enzyme Assays . Orotate PRTase and OMP decarboxylase activities were measured with the standard ~adiotracer assays described previously (5) . One unit of enzyme activity is that amount which catalyzes the removal of 1 nmol of substrate per hr . Sucrose Gradient Centrifugation . Sucrose gradient centrifugation 'of cellfree extracts was performed as before (5) except that the 5 to 20$ sucrose gradients were prepared with a Beckman Gradient Former and fractionated with an upward flow multi-component system from Instrumentation Specialties Co ., Lincoln, Nebraska . Ovalbumin could not be used as an internal protein marker with liver preparations since its absorbance was masked by endogenous liver proteins . No such difficulty was encountered with brain preparations . Internal protein markers were used to calculate the sedimentation coefficients of orotate PRTase and OMP decarboxylase by the method outlined previously (5) .
Vol . 22, No . 7, 1978
Coordinate Behavior of Pyrimidine Enzymaa
57 9
RESULTS AND DISCUSSION Data on the specific activities of orotate PRTase and OMP decarboxylase during mouse development are summarized in Figs .lA and 1B for liver and brain, respectively . Both tissues exhibited a sharp decline in enzyme specific activities from about the time of birth to 21 days of age . Following this initial decline, specific activity values in liver showed a moderate rise and then changed only slightly with further increase in age . A similar pattern was seen in brain except for the absence of any appreciable rise in specific activities immediately following day 21 . The low specific activity values in liver at 21 days of age might reflect the fact that mouse pups were weaned on day 20 . Conceivably, weaning could have caused a temporary nutritional deficit leading to a preferential lowering of liver enzyme levels . Also, the manner in which enzyme specific activities changed during fetal development differed in liver and brain . Liver exhibited a marked increase in specific activities whereas a decrease combined with a relatively small increase at about the time of birth was seen in brain . The point we wish to emphasize concerning the above results is the coordinate behâvior exhibited by orotate PRTase and OMP decarboxylase during liver and brain development . This is evident from the closely parallel nature of the specific activity curves (Figs . lA and 1B) and in a more quantitative manner from the nearly constant ratio of enzyme specific activities (Table I) . TABLE I Relative Activities of Orotate PRTase and OMP Decarboxylase in Developing Mouse Liver and Brain
Age of mice (days)
a
OMP decarboxyl ase/o rotate PRTasea Liver Brain
-8
1 .19
0 .98
-1
1 .08
1 .01
1
1 .27
1 .13
3
1 .13
1 .15 .
10
1 .37
1 .22
21
1 .31
1 .01
35
1 .39
1 .08
63
1 .25
1 .28
90
1 .29
0 .93
Ratio of specific activities (data from Figs . 1B .)
lA and
580
Coorâiaate Behavior of P,yrimidine Easymea I
Vol . 22, No . 7, 1978 ~
I
A. Liver
0
Age (days)
~
I
~
I
so Age (days)
20
4o
~
I
eo
FIG . 1 Specific activities of orotate PRTase and 0I4P decarboxylase in developing mouse liver (A) and brain (B) .
0 Top
02
0.4
Q6
Q8
Fraction of Gradient
L0 Bottom
0
~
0 ToP
FIG. 2
0.2
0.4
i 'R+oiemab y
0.6
0.8
L0
Fraction of Gradient
Sucrose gradient centrifugation of orotate PRTase and 0I~ decarboxylase from fetal mouse liver (A) and brain (B) . Cell-free extracts served as the source of the above enzymes and were prepared from fetuses taken about 8 days prior to birth . The recovery of orotate PRTase and OMP decarboxylase activities in the above experiments was about 45~ and 65~, respectively .
Dol . 22, No, 7, 1978
Coordinate Behavior of Pyrimidine Eazymea
58 1
These results support the view that orotate PRTase and OMP decarboxylase exist as a bifunctional enzyme complex not only in adult but also in immature, neonatal and fetal mouse liver and brain . Further support for this view was provided by the finding that these two enzymes routinely cosedimented during sucrose gradient centrifugation, regardless of the stage of liver or brain development . Figs . 2A and 2B, representative of typical experiments, illustrate the cosedimentation behavior of these two enzymes in cell-free extracts from fetal liver and brain, respectively . Additional support for the existence of a bifunctional enzyme complex has come from enzyme purification studies to be described in detail elsewhere . These studies revealed that orotate PRTase and OMP decarboxylase cochromatograph on Sephadex G-150 and DEAF Bio-Gel A . Evidence that the physical nature of the enzyme complex is similar if not identical in fetal, neonatal, immature and adult tissues was provided by the fact that the sedimentation coefficient of the complex remains essentially constant throughout development (Table II) . It is clear, however, that the sedimentation coefficient of the liver complex (5 .0 t 0 .2) differs from that of the brain complex (4 .3 ± 0 .1) . TABLE II Sedimentation Coefficient of Orotate PRTase-OMP Decarboxylase Complex in Developing Mouse Liver and Brain
Age of mice (days)
Sedimentation coefficient (sec x 10 13 )a Brain Liver
-8
4 .8
4 .2
-1
-
-
1
5 .0
4 .2
3
-
4 .4
10
5 .1
-
21
4 .8
4 .3
35
4 .8
-
63
5 .2
4 .3
90
5 .1
4 .2
a The mean t S .D . of these values was calculated to be 5 .0 f 0 .2 and 4 .3 t 0 .1 for the liver and brain complex, respectively . It must be emphasized that the above results reflect the behavior of orotate PRTase and OMP decarboxylase in fresh cell-free extracts . We have recently noted, however, that multiple molecular forms of these two enzymes
582
Coordiaate Behavior of Pyrimidiae Enzymes
Vol . 22, No . 7, 1978
begin to appear when aged cell-free extracts are employed . Both dissociation and aggregation of the enzyme complex appears to take place in aged preparations . Similar observations have been made by others recently during a study of these two enzymes from human erythrocytes (14) and liver (15) . ACKNOWLEDGMENT This work was supported by United ~tates Public Service Grant CA-15036 from the National Cancer Institute . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
S .H . APPEL, J . Biol . Chem . 243, 3924-3929 (1968) . D . HATFIELD, ârtd J .B . WYNGAARDEN, J . Biol . Chem . 239, 2580-2586 (1964) . D .K . KASKEBAR, A . NAGABHUSHANAM, and D .M . GREENBERG, _J . Biol . Chem. _293, 4245-4249 (1964) . P .R . KAVIPURAPU, and M .E . JONES, J . Biol . Chem . 251, 5589-5599 (1976) . P . REYES, and M .E . GUGANIG, J . Biôl . Chem . 250, 5097-5108 (1975) . G .K . BROWN, R .M . FOX, and W .J . O'SULLIVAN, J.Biol . Chem . 250, 7352-7358 (1975) . M .J . SWEENEY, J .W . PARTON, and D .H . HOFFMAN, Advan . Enzyme Regul . _12, 385-396 (1974) . J . PAUSCH, D . KEPPLER, and K . DECKER, Biochem . Biophys . Acta _258, 395403 (1972) . G .K . BROWN, R .M . FOX, and W .J . O'SULLIVAN, Biochem . Pharmacol . _21, 24692477 (1972) . R .M . FOX, M .H . WOOD, and W .J . O'SULLIVAN, J . Clin . Invest . 50, 1050-1060 (1971) . W .J .M . TAX, J .H . VEERKAMP, and J .M .F . TRIJBELS, Comp . Biochem . Physiol . 54B, 209-212 (1976) . .D . BEARDMORE, J .S . CASHMAN, and W .N . KELLEY, J . Clin . Invest . 51, 1823T 1832 (1972) . O .H . LOWRY, N .J . ROSEBROUQ-i, A .L . FARR, and R .J . RANDALL, _J . Biol . Chem . 193, 265-275 (1951) . .K . BROWN, and W .J . O'SULLIVAN, Biochemistry 16, 3235-3242 (1977) . G M .T . CAMPBELL, N .D . GALLAGHER, and W .J . O'SULLIVAN, Biochem . _Med . _17, 128-140 (1977) .
Lifn Sciences, Vol . 22, pp . 577-582 Printed in the U .S .A .
COORDINATE BEHAVIOR OF OROTATli PHOSPHORIHOSYLTRANSFERASE AND OROTIDYLATE DECARBOXYLASE IN DEVELOPING MOUSE LIVER AND BRAIN Philip Reyesl and Clare Intress Department of Biochemistry The University of New Mexico School of Medicine Albuquerque, New Mexico 87131 (Rrcaived in final form December 19, 1977) SUMMARY A large body of data has accumulated in recent years supporting the view that orotate phosphoribosyltransferase and orotidylate decarboxylase exist as a bif~ctional enzyme complex in adult mamma lian tissues . This paper presents evidence that such a complex also occurs in mouse liver and brain, regardless of the developmental stage of the animal . Orotate phosphoribosyltransferase and orotidylate decarboxylase activities remained coordinate in fetal, neonatal, immature and adult liver and brain . In addition, these two enzymes routinely cosedimented during centrifugation of cell-free extracts in sucrose gradients . The sedimentation coefficient of the enzyme complex did not change significantly during mouse development . However, the liver complex exhibited a sedimentation coefficient (5 .0 t 0 .2) that differed from that of the brain complex (4 .3 t 0 .1 .) INTRODUCTION It is now well established that the de novo synthesis of UMP involves the sequential action of orotate PRTase2 (EC 2 .4 .2 .10) (reaction 1) and OMP decarboxylase (EC 4 .1 .1 .23) (reaction 2 .) 2+ orotate + PP-ribose-P ,~- OMP + PPi (1) OMP
UMP + C02
(2)
An interesting feature of these two enzymes is their capacity to copurify from all mammalian tissues so far examined . These tissues include bovine brain (1), erythrocytes (2) and thymus (3), Ehrlich ascites carcinoma (4), marine leukemia P1534J (5) and human erythrocytes (6) . In addition, the activities of the above enzymes show a coordinate relationship in rat (7-9) and human (10) tissues and in erythrocytes from various mammalian species (11) . It is also well established that administration to rats and humans of the xanthine oxidase inhibitor, allopurinol (4-hydroxypyrazolo (3,4-d) pyrimidine), produces a coordinate increase in the activities of both enzymes (9,10,12) . 1. 2.
To whom reprint requests should be addressed . Abbreviations : PRTase, phosphoribosyltransferase ; OMP, orotidine 5'-monophosphate ; PP-ribose-P, 5-phosphoribosyl 1-pyrophosphate . 577
578
Coordinate Behavior of Pyrimidine Enzymaa
Vol . 22, No . 7, 1978
In view of the above findings, it is now generally believed that orotate PRTase and OMP decarboxylase exist as a bifunctional enzyme complex in mammals . It is to be noted, however, that all previous studies of this complex have emphasized the use of adult tissues . It was therefore of interest to examine fetal, neonatal, immature as well as adult mouse liver and brain in order to gather data on the occurrence of this enzyme complex during growth and development . An equally important goal of this study was to quantitate the level of activity of these two enzymes in developing mouse liver and brain . MATERIALS AND METHODS Animals . CD2Fl hybrid mice were used throughout this study . These mice representtree offspring of BALB/c inbred females mated with DBA/2 inbred males . Pregnant BALB/c mice, obtained from Lab Supply Co ., Indianapolis, Indiana, were caged individually, fed ad libitum , and housed in a room illuminated from 9 p .m . to 9 a .m . Gestationalâge was estimated retrospectively based on the day of birth of the remaining litters, all of which were born within a period of 2 days . The error in gestational age is therefore about t 1 day . Mice were weaned at 20 days of age and were segregated according to sex (6 mice per cage) 35 days after birth . Experimental data points on day -8, -1, 1, 3, 10, 21 and 35 were each obtained with pooled brains and livers from an entire litter (average of 9 animals per litter) whereas 3 males and 3 females (not necessarily from the same litter) were used as the source of pooled tissues on days 63 and 90 . Preparation of Cell-free Extracts . All animals were routinely sacrificed by decapitation during the last 30 min of the light period . Brains and livers were excised quickly, rinsed with ice-cold 0 .15 M NaCl, gently bottled, weighed and minced . In the case of pregnant mice, the uterus was removed in toto following which the fetuses were immediately delivered . All subsequent steps were done at 0-5 ° . The minced tissues were then suspended in 5 volumes of 50 mM potassium phosphate (pH 8 .0) containing 1 mM'PP-ribose-P and 1 mM dithiothreitol . Cells were broken with 7 strokes of a glass-Teflon homogenizer . The resulting suspension was centrifuged at 105,000 x g for 60 min and the supernatant fraction removed with as little of the overlying lipid as possible . This fraction, which served as the source of orotate PRTase and OMP decarboxylase, was normally assayed for enzyme activity following overnight storage at -20 ° . Analysis of this fraction by sucrose gradient centrifugation was usually done within a week of its preparation . Protein concentrations were determined by the method of Lowry et al . (13), with bovine serum albumin as standard . Enzyme Assays . Orotate PRTase and OMP decarboxylase activities were measured with the standard ~adiotracer assays described previously (5) . One unit of enzyme activity is that amount which catalyzes the removal of 1 nmol of substrate per hr . Sucrose Gradient Centrifugation . Sucrose gradient centrifugation 'of cellfree extracts was performed as before (5) except that the 5 to 20$ sucrose gradients were prepared with a Beckman Gradient Former and fractionated with an upward flow multi-component system from Instrumentation Specialties Co ., Lincoln, Nebraska . Ovalbumin could not be used as an internal protein marker with liver preparations since its absorbance was masked by endogenous liver proteins . No such difficulty was encountered with brain preparations . Internal protein markers were used to calculate the sedimentation coefficients of orotate PRTase and OMP decarboxylase by the method outlined previously (5) .
Vol . 22, No . 7, 1978
Coordinate Behavior of Pyrimidine Enzymaa
57 9
RESULTS AND DISCUSSION Data on the specific activities of orotate PRTase and OMP decarboxylase during mouse development are summarized in Figs .lA and 1B for liver and brain, respectively . Both tissues exhibited a sharp decline in enzyme specific activities from about the time of birth to 21 days of age . Following this initial decline, specific activity values in liver showed a moderate rise and then changed only slightly with further increase in age . A similar pattern was seen in brain except for the absence of any appreciable rise in specific activities immediately following day 21 . The low specific activity values in liver at 21 days of age might reflect the fact that mouse pups were weaned on day 20 . Conceivably, weaning could have caused a temporary nutritional deficit leading to a preferential lowering of liver enzyme levels . Also, the manner in which enzyme specific activities changed during fetal development differed in liver and brain . Liver exhibited a marked increase in specific activities whereas a decrease combined with a relatively small increase at about the time of birth was seen in brain . The point we wish to emphasize concerning the above results is the coordinate behâvior exhibited by orotate PRTase and OMP decarboxylase during liver and brain development . This is evident from the closely parallel nature of the specific activity curves (Figs . lA and 1B) and in a more quantitative manner from the nearly constant ratio of enzyme specific activities (Table I) . TABLE I Relative Activities of Orotate PRTase and OMP Decarboxylase in Developing Mouse Liver and Brain
Age of mice (days)
a
OMP decarboxyl ase/o rotate PRTasea Liver Brain
-8
1 .19
0 .98
-1
1 .08
1 .01
1
1 .27
1 .13
3
1 .13
1 .15 .
10
1 .37
1 .22
21
1 .31
1 .01
35
1 .39
1 .08
63
1 .25
1 .28
90
1 .29
0 .93
Ratio of specific activities (data from Figs . 1B .)
lA and
580
Coorâiaate Behavior of P,yrimidine Easymea I
Vol . 22, No . 7, 1978 ~
I
A. Liver
0
Age (days)
~
I
~
I
so Age (days)
20
4o
~
I
eo
FIG . 1 Specific activities of orotate PRTase and 0I4P decarboxylase in developing mouse liver (A) and brain (B) .
0 Top
02
0.4
Q6
Q8
Fraction of Gradient
L0 Bottom
0
~
0 ToP
FIG. 2
0.2
0.4
i 'R+oiemab y
0.6
0.8
L0
Fraction of Gradient
Sucrose gradient centrifugation of orotate PRTase and 0I~ decarboxylase from fetal mouse liver (A) and brain (B) . Cell-free extracts served as the source of the above enzymes and were prepared from fetuses taken about 8 days prior to birth . The recovery of orotate PRTase and OMP decarboxylase activities in the above experiments was about 45~ and 65~, respectively .
Dol . 22, No, 7, 1978
Coordinate Behavior of Pyrimidine Eazymea
58 1
These results support the view that orotate PRTase and OMP decarboxylase exist as a bifunctional enzyme complex not only in adult but also in immature, neonatal and fetal mouse liver and brain . Further support for this view was provided by the finding that these two enzymes routinely cosedimented during sucrose gradient centrifugation, regardless of the stage of liver or brain development . Figs . 2A and 2B, representative of typical experiments, illustrate the cosedimentation behavior of these two enzymes in cell-free extracts from fetal liver and brain, respectively . Additional support for the existence of a bifunctional enzyme complex has come from enzyme purification studies to be described in detail elsewhere . These studies revealed that orotate PRTase and OMP decarboxylase cochromatograph on Sephadex G-150 and DEAF Bio-Gel A . Evidence that the physical nature of the enzyme complex is similar if not identical in fetal, neonatal, immature and adult tissues was provided by the fact that the sedimentation coefficient of the complex remains essentially constant throughout development (Table II) . It is clear, however, that the sedimentation coefficient of the liver complex (5 .0 t 0 .2) differs from that of the brain complex (4 .3 ± 0 .1) . TABLE II Sedimentation Coefficient of Orotate PRTase-OMP Decarboxylase Complex in Developing Mouse Liver and Brain
Age of mice (days)
Sedimentation coefficient (sec x 10 13 )a Brain Liver
-8
4 .8
4 .2
-1
-
-
1
5 .0
4 .2
3
-
4 .4
10
5 .1
-
21
4 .8
4 .3
35
4 .8
-
63
5 .2
4 .3
90
5 .1
4 .2
a The mean t S .D . of these values was calculated to be 5 .0 f 0 .2 and 4 .3 t 0 .1 for the liver and brain complex, respectively . It must be emphasized that the above results reflect the behavior of orotate PRTase and OMP decarboxylase in fresh cell-free extracts . We have recently noted, however, that multiple molecular forms of these two enzymes
582
Coordiaate Behavior of Pyrimidiae Enzymes
Vol . 22, No . 7, 1978
begin to appear when aged cell-free extracts are employed . Both dissociation and aggregation of the enzyme complex appears to take place in aged preparations . Similar observations have been made by others recently during a study of these two enzymes from human erythrocytes (14) and liver (15) . ACKNOWLEDGMENT This work was supported by United ~tates Public Service Grant CA-15036 from the National Cancer Institute . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
S .H . APPEL, J . Biol . Chem . 243, 3924-3929 (1968) . D . HATFIELD, ârtd J .B . WYNGAARDEN, J . Biol . Chem . 239, 2580-2586 (1964) . D .K . KASKEBAR, A . NAGABHUSHANAM, and D .M . GREENBERG, _J . Biol . Chem. _293, 4245-4249 (1964) . P .R . KAVIPURAPU, and M .E . JONES, J . Biol . Chem . 251, 5589-5599 (1976) . P . REYES, and M .E . GUGANIG, J . Biôl . Chem . 250, 5097-5108 (1975) . G .K . BROWN, R .M . FOX, and W .J . O'SULLIVAN, J.Biol . Chem . 250, 7352-7358 (1975) . M .J . SWEENEY, J .W . PARTON, and D .H . HOFFMAN, Advan . Enzyme Regul . _12, 385-396 (1974) . J . PAUSCH, D . KEPPLER, and K . DECKER, Biochem . Biophys . Acta _258, 395403 (1972) . G .K . BROWN, R .M . FOX, and W .J . O'SULLIVAN, Biochem . Pharmacol . _21, 24692477 (1972) . R .M . FOX, M .H . WOOD, and W .J . O'SULLIVAN, J . Clin . Invest . 50, 1050-1060 (1971) . W .J .M . TAX, J .H . VEERKAMP, and J .M .F . TRIJBELS, Comp . Biochem . Physiol . 54B, 209-212 (1976) . .D . BEARDMORE, J .S . CASHMAN, and W .N . KELLEY, J . Clin . Invest . 51, 1823T 1832 (1972) . O .H . LOWRY, N .J . ROSEBROUQ-i, A .L . FARR, and R .J . RANDALL, _J . Biol . Chem . 193, 265-275 (1951) . .K . BROWN, and W .J . O'SULLIVAN, Biochemistry 16, 3235-3242 (1977) . G M .T . CAMPBELL, N .D . GALLAGHER, and W .J . O'SULLIVAN, Biochem . _Med . _17, 128-140 (1977) .