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Coenzyme A and diphosphopyridine nucleotide in guinea pig mammary tissue
Coenzyme A and Diphosphopyridine Nucleotide in Guinea Pig Mammary Tissue” Ira Ringler, Nonvin From
the Department
Becker and Walter L. Nelson
of Biochemistry and Nutrition and The Cornell University, Ithaca, New York Received
March
School
of Nutrition,
1, 1954
INTRODUCTION In a previous paper, Moore and Nelson (1) reported striking increases in the activity of certain oxidative enzymes when the metabolism of mammary gland tissue shifted from growth to lactation. This observat,ion led to the investigation of other metabolically important factors which might influence the shift in enzymatic activity. This report deals with the measurement of coenzyme A and diphosphopyridine nucleotide (DPN) concentrations at various stages of development in the mammary glands of guinea pigs. Also included are observations on the intracellular distribution of these cofactors, as well as some brief comments on the enzymatic determination of coenzyme A concentration in mammary gland tissue. EXPERIMENTAL All animals were maintained on a stock diet of Rockland Farm guinea pig pellets plus timothy hay, fresh greens, and water. In studies with lactating animals, the young were allowed to suckle until the time of sacrifice. Animals were sacrificed by a blow on the head and exsanguination. The mammary tissue was quickly removed, weighed, and placed in a cold all-glass homogenizer (2) containing sutlicient phosphate buffer (0.1 M) pH 7.4 to prepare a 10% homogenate. One milliliter of this homogenate was used to determine dry weight, and for lactating mammary tissue, corrections were made for milk content (1). Aliquots of the chilled homogenate were added to prepared tubes at 0.05-, O.lO-, 0.15., and 0.20.ml. levels for the coenzyme A assay. Coenzyme A was determined by the sulfanilamide acetylation method of Kaplan and Lipmann, and reported in Lipmann units per gram dry weight, and, in one experiment, per gram wet weight (3). DPN was measured spectrophotometrically by a previously described method (4). 1 This study was supported Inc., New York, N. Y.
in part
by a grant 348
from
the Nutrition
Foundation,
COENZYME
h AND DPS
3-M
The preparation anddifferential centrifugationof the mammarygland homogenates was carried out in 0.1 M phosphate buffer, pH 7.4. The nuclear fraction was collected at 600 X 9 for 30 min. in a Universal PR-1 refrigerated centrifuge, employing two washings. The mitochondria-microsome fraction was collect,ed at 1-1-I,000 X q, using a Spinco ultracentrifuge, for 1 hr., employing one washing. The final washing was combined with the supernatant for assay.
RESULTS
AND DISCUSSION
The data presented in Table I illustrate the changes in coenzyme A concentration in the guinea pig mammary gland during growth, lactation, and involution. During pregnancy, as indicated by fetal size, the coenzyme A level remained low but began to rise 24 hr. post partum, reaching a maximum at about the 5th day post partum. After this pronounced initial rise, the concentration declined at a much slower rate. These changes in coenzyme A follow t’he general trend of milk production in guinea pigs, which reaches a maximum 2-3 days after parturition (-5). Low coenzyme values in somelactating animals may he accounted TABLE Cocnzyme Point
A Content
in the gestation-lactation
2 in. fetus 2% in. fetus 336 in. fetus 4 in. fetus 1 day post 2 days post 3 days post 4 days post 5 days post 6 days post 8 days post 12 days post 13 days post 22 days post 26 days post 27 days post 31 days post 41 days post 42 days post 46 days post 49 days post a Ii:nch
coenzyme
A value
I
of Guinea cycle
Pi!\ Lipmann
units/g.
22 30 23 26, 55 74 101, 105 170 165, 131 126 86 73 06 73 64 27 47 41 40
partum partum partum partum partum partum partum partum partum partum partum partum partum partum partum partum partum
represents
Mnmmary
mammary
Tissue dry weight
tissueQ
47
120,
99
159,
80,
tissue
from
I43
one animal.
350
RINGLER,
BECKER
AND
NELSON
for in their failure to lactate properly. This was noted in several cases upon dissection of the mammary gland. The DPN concentration in tissue homogenates was determined concurrently with coenzyme A for ten animals during various stages of gestation and lactation. The results indicated that the DPN concentration (micrograms per gram wet weight) remained relatively constant through gestation, average 204 (range 156-266) and lactation, average 268 (range 212-304). The intracellular distribution of coenzyme A and DPN in two lactating and two nonlactating mammary glands was determined. Since the distribution was similar in both cases, the values for one determination only are presented in Table II. In general, the intracellular distribution of both coenzymes remains relatively the same in the different physiological states of this tissue. In mammary tissue approximately 50 % of the coenzyme A was found in the supernatant fraction, the remaining units being evenly distributed in the nuclear and mitochondria-microsome fractions. In contrast to this, Higgins et al. (6) reported that for rat liver over 50% of the coenzyme was found in the mitochondria. However, a notable similarity does exist between the intracellular distribution of coenzyme A in liver tumors (6) and mammary tissue. The intracellular distribution of DPN was similar to that of coenzyme A. Over 50 % of the DPN was assayed in the supernatant, the remaining concentrations being evenly distributed in the nuclear and mitochondriamicrosome fractions. TABLE Zntracellular
Distribution
II
of Coenzyme A and Guinea Pig Mammary Coenzyme
I
Diphosphopyridine Tissue
Aa
a Lipmann units/g. fresh tissue. b Micrograms/g. fresh tissue.
49 6 29 9 44
DPNb
I
Nonlactating tissue
Whole homogenate Nuclei Supernatant Mitochondria-microsome Recovery
Nucleotide
21 6 10 7 23
I-y;;:”
299 80 180 82 342
Nonlactating tissue
266 58 149 86 293
in
COENZYME
A
.4ND
DPN
351
TABLE III A
Comparison
of the Coenzyme A Concentration
= I
Homogenates
Lactating Lactating Lact:tting Lactat,ing Xonlactating a Lipmann b Lipmann
units/g. units/g.
I ~
before
and after
in Guinea Pig Heat Treatment
Coenzyme
101 126 114
26
T1 127 115
23
Mammary
Tissue
A concentration
105
156
-
dry weight. fresh weight.
Previous studies with liver tissue indicated the necessity to heat, homogenates before determining its coenzyme ii content (3). although mammary homogenat8eswere found active when kept cold and used directly for assay, it was not known whet)her the act’ivity equaled that of heat-inactivated homogenates. A comparative study was t’herefore conducted to determine the relative coenzyme activity in mammary tissue, wit,h and without previous heat treatment. Homogenates were prepared as described; one aliquot was assayed directly, while another portion was heated for 3 min. in a boiling water bath, rehomogenized, and assayed. Liver was similarly assayed for comparison. From the results given in Table III, it is evident that there is little difference in the roenzyme A activity in mammary t’issue, with and without heat inactivat~ion. These data indicate that the enzymes responsible for the) breakdown of coenzyme A are either absent or present in very low concsent,rationin guinea pig mammary tissue.
SUMMARY The coenzyme A and diphosphopyridine nucleotide (DPK) concentrations in guinea pig mammary gland tissue during gestation, lactation, and involution are reported. DPN remains relatively constant through growth and lactation, while the coenzyme A content is lowest during the period of growth, highest following parturition. The intracellular distribution of coenzyme A and DPN in lactating and nonlactating mammary gland tissue is similar, the highest concentration being found in
352
RINGLER,
BECKER
AND
NELSON
the supernatant fraction. Heat inactivation of mammary coenzyme A determination was found unnecessary.
tissue before
REFERENCES 1. 2. 3. 4.
MOORE, R. O., AND NELSON, W. L., Arch. Biochem. and Biophys. 36,178 (1952). POTTER, V. R., AND ELVEHJEM, C. A., J. Viol. Chem. 114,495 (1936). KAPLAN, N. O., AND LIPMANN, F., J. Biol. Chem. 174, 37 (1948). STRENGTH, D. R., RINGLER, I., AND NELSON, W. L., Arch. Biochem. and Biophys. 48, 107 (1954). 5. NELSON, W. L., KAYE, A., MOORE, M., WILLIAMS, H. H., AND HERRINGTON, B. L., J. Nutrition 44, 585 (1951). 6. HIGGINS, H., MILLER, J. A., PRICE, J. M., AND STRONG. F. M., Proc. Sot. Ezptl. Biol. Med. 76, 462 (1950).
the Department
Becker and Walter L. Nelson
of Biochemistry and Nutrition and The Cornell University, Ithaca, New York Received
March
School
of Nutrition,
1, 1954
INTRODUCTION In a previous paper, Moore and Nelson (1) reported striking increases in the activity of certain oxidative enzymes when the metabolism of mammary gland tissue shifted from growth to lactation. This observat,ion led to the investigation of other metabolically important factors which might influence the shift in enzymatic activity. This report deals with the measurement of coenzyme A and diphosphopyridine nucleotide (DPN) concentrations at various stages of development in the mammary glands of guinea pigs. Also included are observations on the intracellular distribution of these cofactors, as well as some brief comments on the enzymatic determination of coenzyme A concentration in mammary gland tissue. EXPERIMENTAL All animals were maintained on a stock diet of Rockland Farm guinea pig pellets plus timothy hay, fresh greens, and water. In studies with lactating animals, the young were allowed to suckle until the time of sacrifice. Animals were sacrificed by a blow on the head and exsanguination. The mammary tissue was quickly removed, weighed, and placed in a cold all-glass homogenizer (2) containing sutlicient phosphate buffer (0.1 M) pH 7.4 to prepare a 10% homogenate. One milliliter of this homogenate was used to determine dry weight, and for lactating mammary tissue, corrections were made for milk content (1). Aliquots of the chilled homogenate were added to prepared tubes at 0.05-, O.lO-, 0.15., and 0.20.ml. levels for the coenzyme A assay. Coenzyme A was determined by the sulfanilamide acetylation method of Kaplan and Lipmann, and reported in Lipmann units per gram dry weight, and, in one experiment, per gram wet weight (3). DPN was measured spectrophotometrically by a previously described method (4). 1 This study was supported Inc., New York, N. Y.
in part
by a grant 348
from
the Nutrition
Foundation,
COENZYME
h AND DPS
3-M
The preparation anddifferential centrifugationof the mammarygland homogenates was carried out in 0.1 M phosphate buffer, pH 7.4. The nuclear fraction was collected at 600 X 9 for 30 min. in a Universal PR-1 refrigerated centrifuge, employing two washings. The mitochondria-microsome fraction was collect,ed at 1-1-I,000 X q, using a Spinco ultracentrifuge, for 1 hr., employing one washing. The final washing was combined with the supernatant for assay.
RESULTS
AND DISCUSSION
The data presented in Table I illustrate the changes in coenzyme A concentration in the guinea pig mammary gland during growth, lactation, and involution. During pregnancy, as indicated by fetal size, the coenzyme A level remained low but began to rise 24 hr. post partum, reaching a maximum at about the 5th day post partum. After this pronounced initial rise, the concentration declined at a much slower rate. These changes in coenzyme A follow t’he general trend of milk production in guinea pigs, which reaches a maximum 2-3 days after parturition (-5). Low coenzyme values in somelactating animals may he accounted TABLE Cocnzyme Point
A Content
in the gestation-lactation
2 in. fetus 2% in. fetus 336 in. fetus 4 in. fetus 1 day post 2 days post 3 days post 4 days post 5 days post 6 days post 8 days post 12 days post 13 days post 22 days post 26 days post 27 days post 31 days post 41 days post 42 days post 46 days post 49 days post a Ii:nch
coenzyme
A value
I
of Guinea cycle
Pi!\ Lipmann
units/g.
22 30 23 26, 55 74 101, 105 170 165, 131 126 86 73 06 73 64 27 47 41 40
partum partum partum partum partum partum partum partum partum partum partum partum partum partum partum partum partum
represents
Mnmmary
mammary
Tissue dry weight
tissueQ
47
120,
99
159,
80,
tissue
from
I43
one animal.
350
RINGLER,
BECKER
AND
NELSON
for in their failure to lactate properly. This was noted in several cases upon dissection of the mammary gland. The DPN concentration in tissue homogenates was determined concurrently with coenzyme A for ten animals during various stages of gestation and lactation. The results indicated that the DPN concentration (micrograms per gram wet weight) remained relatively constant through gestation, average 204 (range 156-266) and lactation, average 268 (range 212-304). The intracellular distribution of coenzyme A and DPN in two lactating and two nonlactating mammary glands was determined. Since the distribution was similar in both cases, the values for one determination only are presented in Table II. In general, the intracellular distribution of both coenzymes remains relatively the same in the different physiological states of this tissue. In mammary tissue approximately 50 % of the coenzyme A was found in the supernatant fraction, the remaining units being evenly distributed in the nuclear and mitochondria-microsome fractions. In contrast to this, Higgins et al. (6) reported that for rat liver over 50% of the coenzyme was found in the mitochondria. However, a notable similarity does exist between the intracellular distribution of coenzyme A in liver tumors (6) and mammary tissue. The intracellular distribution of DPN was similar to that of coenzyme A. Over 50 % of the DPN was assayed in the supernatant, the remaining concentrations being evenly distributed in the nuclear and mitochondriamicrosome fractions. TABLE Zntracellular
Distribution
II
of Coenzyme A and Guinea Pig Mammary Coenzyme
I
Diphosphopyridine Tissue
Aa
a Lipmann units/g. fresh tissue. b Micrograms/g. fresh tissue.
49 6 29 9 44
DPNb
I
Nonlactating tissue
Whole homogenate Nuclei Supernatant Mitochondria-microsome Recovery
Nucleotide
21 6 10 7 23
I-y;;:”
299 80 180 82 342
Nonlactating tissue
266 58 149 86 293
in
COENZYME
A
.4ND
DPN
351
TABLE III A
Comparison
of the Coenzyme A Concentration
= I
Homogenates
Lactating Lactating Lact:tting Lactat,ing Xonlactating a Lipmann b Lipmann
units/g. units/g.
I ~
before
and after
in Guinea Pig Heat Treatment
Coenzyme
101 126 114
26
T1 127 115
23
Mammary
Tissue
A concentration
105
156
-
dry weight. fresh weight.
Previous studies with liver tissue indicated the necessity to heat, homogenates before determining its coenzyme ii content (3). although mammary homogenat8eswere found active when kept cold and used directly for assay, it was not known whet)her the act’ivity equaled that of heat-inactivated homogenates. A comparative study was t’herefore conducted to determine the relative coenzyme activity in mammary tissue, wit,h and without previous heat treatment. Homogenates were prepared as described; one aliquot was assayed directly, while another portion was heated for 3 min. in a boiling water bath, rehomogenized, and assayed. Liver was similarly assayed for comparison. From the results given in Table III, it is evident that there is little difference in the roenzyme A activity in mammary t’issue, with and without heat inactivat~ion. These data indicate that the enzymes responsible for the) breakdown of coenzyme A are either absent or present in very low concsent,rationin guinea pig mammary tissue.
SUMMARY The coenzyme A and diphosphopyridine nucleotide (DPK) concentrations in guinea pig mammary gland tissue during gestation, lactation, and involution are reported. DPN remains relatively constant through growth and lactation, while the coenzyme A content is lowest during the period of growth, highest following parturition. The intracellular distribution of coenzyme A and DPN in lactating and nonlactating mammary gland tissue is similar, the highest concentration being found in
352
RINGLER,
BECKER
AND
NELSON
the supernatant fraction. Heat inactivation of mammary coenzyme A determination was found unnecessary.
tissue before
REFERENCES 1. 2. 3. 4.
MOORE, R. O., AND NELSON, W. L., Arch. Biochem. and Biophys. 36,178 (1952). POTTER, V. R., AND ELVEHJEM, C. A., J. Viol. Chem. 114,495 (1936). KAPLAN, N. O., AND LIPMANN, F., J. Biol. Chem. 174, 37 (1948). STRENGTH, D. R., RINGLER, I., AND NELSON, W. L., Arch. Biochem. and Biophys. 48, 107 (1954). 5. NELSON, W. L., KAYE, A., MOORE, M., WILLIAMS, H. H., AND HERRINGTON, B. L., J. Nutrition 44, 585 (1951). 6. HIGGINS, H., MILLER, J. A., PRICE, J. M., AND STRONG. F. M., Proc. Sot. Ezptl. Biol. Med. 76, 462 (1950).