Regulation of Volatile Fatty Acid Uptake by Mitochondrial Acyl CoA Synthetases of Bovine Heart1, 2

Regulation of Volatile Fatty Acid Uptake by Mitochondrial Acyl CoA Synthetases of Bovine Heart 1,2 C A T H E R I N E A. RICKS 3 and R, M. COOK Department of Dairy Science Michigan State University East Lansing 48824

synthetases that are necessary for tissue uptake and subsequent metabolism of ruminally derived volatile fatty acids in heart, kidney, and liver mitochondria of lactating Holstein cows. Campagnari and Webster (3) using acetate as a substrate to monitor enzyme activity isolated a distinct acetyl CoA synthetase activating both acetate and propionate from beef heart mitochondria. Similar enzymes have been isolated from goat mammary mitochondria (6) and from bovine mammary mitochondria (14). Webster et al. (26) using butyrate as a substrate to measure enzyme activity reported a butyrate-activating enzyme in bovine heart mitochondria. This enzyme differs from the butyryl CoA synthetase purified by Mahler et al. (10) and by Ricks and Cook (18) from bovine liver. Our experiments were to obtain a complete profile of forms of these enzymes in heart mitochondria. In view of overlapping substrate specificities of acyl CoA synthetases in ruminants (17), it was essential to monitor all fractions during enzyme purification with acetate, propionate, and butyrate as substrates for detecting enzymatic activity. In addition, characterization studies on purified enzyme preparations were conducted to elucidate how volatile fatty acid uptake by heart mitochondria is controlled.

A BST R ACT

Purification of components of heart mitochondria activating short chain fatty acids prepared from tissue of lactating Holstein cows demonstrated predominandy one acyl CoA synthetase, acetyl CoA synthetase activating acetate, and propionate. Activity of butyryl CoA synthetase was low. Propionyl CoA synthetase characteristically in bovine liver and kidney tissue could not be demonstrated in heart mitochondria. Thus, of the ruminally derived volatile fatty acids only acetate can be used by heart mitochondria as a primary energy source because of small quantities of propionate in peripheral blood. Acetyl CoA synthetase was a glycoprotein composed of a single polypeptide chain of apparent molecular weight 67,500. The Michaelis-Menten constant for acetate was 1.8 x 10-4M. By comparison with literature for blood acetate concentration we concluded that enzyme is saturated with substrate at all physiological concentrations of acetate. These kinetic properties ensure a constant supply of acetate as an energy source for maintaining heart function in ruminants. INTRODUCTION

This and companion papers describe purification of a series of enzymes termed acyl CoA

Received May 2, 1980. 1Published with approval of the Director of the Agricultural Experiment Station as Journal Article No. 9404. 2Data taken from dissertation by the senior author in partial fulfillment of the requirements for the Ph.D. degree. 3American Cyanamid Company, P. O. Box 400, Princeton, NJ 08540. 1981 J Dairy Sci 64:2336--2343

M A T E R I A L S A N D METHODS Enzyme Assay

Enzyme activity was determined as described by Quraishi and Cook (13); each fraction was assayed for its ability to activate acetate, propionate, and butyrate. One unit of enzyme is defined as the amount that catalyzes the disappearance of 1 nmol of coenzyme A per minute. Protein Determination

23 36

Protein was measured by the procedure of

BOVINE HEART ACYL CoA SYNTHETASES

233 7

Enzyme Characterization

Purified enzyme was assayed for carbohydrate residues by gas chromatographic analysis of trimethylsilyl derivatives as described by Stamoudis (22). Sialic acid content of purified protein was determined by the method of Warren (24). The enzyme used for these latter two analyses was purified in the absence of glycerol. Michaelis-Menten constants (Kin) and maximal velocities (Vmax) were determined from Lineweaver-Burk and Eadie-Hofstee plots by linear regression. Theoretical curves were obtained by Km and Vmax derived from the Eadie-Hofstee plot.

Enzyme purity was assessed by polyacrylamide disc gel electrophoresis as described by Davis (7). After electrophoresis, gels were fixed and strained by the procedure of Chrambach et al. (4). In some cases the periodic acid-Schiff (PAS) staining technique of Hotchkiss (8) for detection of carbohydrate components was used following electrophoresis on acrylamide gels. Molecular weights of purified enzyme preparations were determined by sucrose density centrifugation (12). Subunit molecular weights of purified enzyme protein were determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (Welton and Feigner, personal communication).

Enzymes were liberated from mitochondria by sonication (Table 1). The mitochondrial extract showed maximal activity towards acetate followed by propionate, whereas activity on butyrate and valerate was lower (Table 1). Mitochondria were isolated and ammonium sulfate fractions prepared the same day. The ammonium sulfate precipitate was stored at - 2 0 ° C until required for chromatography. Column chromatography of dialyzed ammonium sulfate fraction on DEAE--23 cellulose is in Figure 1. Enzyme activity could be eluted

Lowry et al. (9). During chromatography the protein content of column effluents was measured by the method of Warburg and Christian (23). Enzyme Purification

Heart tissue was obtained from lactating cows slaughtered at a local abattoir. No data were available on their milking history. Isolation of mitochondria, ammonium sulfate fractionation, DEAE--23 cellulose, and calcium phosphate gel chromatography were as in (18).

R ESU LTS

TABLE 1. Purification of acetyl CoA synthetase from bovine heart mitochondria. The procedure was developed for purification of fatty acid activating enzymes from liver (18).

Fraction

Total protein

Total units C2

(mg) Mitochondrial suspension Mitochondrial extract c Ammonium sulfate precipitate DEAE--23 cellulose Calcium phosphate gel

Specific activityb C2

C3

(units/mg protein)a

Cs

Yield

Fold purification

....

(%)

798

9,490

12

11

8

11

100

1

331

36,438

110

58

39

24

384

9

33

15,246

462

213

71

58

161

39

2,700 573

1,038 9,550

500 4,000

0 0

0 0

29 6

87 796

2.6 0.06

-

C4

aA unit is defined as the amount of enzyme which catalyzes the disappearance of 1 nmol of coenzyme A per min. bc 2 = acetate; C3 = propionate; C4 = butyrate; C4 = valerate. Cprepared by sonication of mitochondria. Amount of heart tissue used = 740 g; wet weight of mitochondria = 8 . 5 g.

Journal of Dairy Science Vol. 64, No. 12, 1981

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RICKS A N D C O O K

synthetase purified by Webster et al. (26). Some butyrate activation was associated with the major enzyme peak. The major enzyme peak was concentrated and applied to a calcium phosphate gel column (Figure 2). The enzyme activating acetate and propionate eluted in the .08 M potassium phosphate buffer. The concentrated enzyme activated primarily acetate followed by propionate with negligible activity on butyrate or valerate (Table 1). Complete purification is in Table 1. A 796 times purification was achieved. The purified enzyme was stable for months

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Figure 1. C h r o m a t o g r a p h y o f the fatty acid activating e n z y m e s of heart m i t o c h o n d r i a on DEAE--23 cellulose. C o l u m n dimensions were 2 cm × 40 cm. The column was washed with 20 ml o f .005 M TrisHCI buffer pH 7.5 followed by 20 mt o f .0i M TrisHCI buffer pH 7.5• Activity was eluted with 100 ml o f a linear KCI gradient o f 0 to .6 M in .01 M Tris-HC1 buffer, pH 7•5. All buffers contained 10% glycerol and 2.5 m M 2-mercaptoethanol. Flow was 40 ml/h. The eluate was collected in 2.3-mi fractions. Collection tubes contained .25 ml glycerol. .. acetate activation; propionate activation; . . . butyrate activation; .. protein• A unit o f enzyme activity is defined as the a m o u n t which catalyzes the disappearance o f 1 n m o l of c o e n z y m e A per minute.

by a KC1 gradient. Both acetate and propionate activating ability coeluted as a single peak. Little

butyrate

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although a small peak eluted in the equilibration buffer at a point where butyrate activation of l i v e r a n d k i d n e y m i t o c h o n d r i a e l u t e d ( 1 8 , 19). This probably represents the butyryl CoA Journal o f Dairy Science Vol. 64, No. 12, 1981

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Figure 2. C h r o m a t o g r a p h y of acetyl CoA synthetase of heart m i t o c h o n d r i a on calcium phosphate gel (with e n z y m e prepared from DEAE--23 cellulose chromatography Figure 1). C o l u m n dimensions were 1.3 cm X 4.6 cm. The column was washed with a stepwise gradient on increasing concentration of potassium p h o s p h a t e buffer, pH 7.0 in 10% glycerol, and 2.5 mM 2-mercaptoethanol. Flow was 20 ml/h. The eluate was collected in 1-ml fractions• Collection tubes contained •1 ml glycerol. - . - • acetate activation; - propionate activation; .. protein•

BOVINE HEART ACVL CoA SYNTHETASES •fit:

ovalbumin

purified enzyme. Results are in Table 2. Mannose, galactose, glucose, n-acetyl galactosamine, and n-acetyl glucosamine were present• Effect of acetate concentration on activity of acetyl CoA synthetase is in Figure 5. The Km was 2.0 x 10--4M by the LineweaverBurk plot (r = .969) and 1.8 x 1 0 - 4 M by the Eadie-Hofstee plot (r = --.945). Enzyme activities with various other substrates are in Table 3. Maximal activity was with acrylate as a substrate followed by acetate and propionate. No activity could be detected with butyrate or valerate as substrates.

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LOG M O L E C U L A R W E I G H T

Figure 3. Sodium dodecyl sulfate polyacrylamide gel eIectrophoresis of purified acetyl CoA synthetase isolated from bovine heart mitochondria and standards of known molecular weight.

DISCUSSION

Procedures to purify acetyl CoA synthetase in the absence of glycerol at - 2 0 ° C . This stability was in contrast to the acyl CoA synthetase purified from liver which denatured in the absence of glycerol (18). The enzyme showed a tendency to aggregate and bind to glass as after storage in glass containers at - 2 0 ° C enzyme protein decreased. Electrophoresis showed one band indicating that the preparation was homogeneous. Electrophoresis followed by the PAS stain was positive indicating the presence of carbohydrate residues. Electrophoresis in the presence of SDS also gave one band. Apparent molecular weight by SDS polyacrylarnide electrophoresis (Figure 3) was 73,000. Molecular weight also was measured by sucrose density centrifugation. Both acetate and propionate were substrates to measure enzyme activity. Enzyme activity was associated with one peak (Figure 4), and this had an apparent molecular weight of 62,000. The enzyme is composed of one polypeptide chain of apparent molecular weight 67,500. The amount of sialic acid was measured in each of three preparations of purified enzyme. Sialic acid contents of the three preparations were .79, 1.92, and 3.72/lg/mg protein. Treatment with sialidase for 30 min at 37°C in acetate buffer at pH 6.0 and subsequent dialysis against Tris-HC1 buffer, pH 8.6, had no effect on enzyme activity when either acetate or propionate were substrates suggesting that sialic acid does n o t influence enzyme activity. Gas liquid chromatography for detection of sugar residues was on six preparations of

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Figure 4. Sucrose density centrifugation of acetyl CoA synthetase of heart mitochondria. Centrifugation was at 50,000 rpm for 12 h at 40°C with a 5 to 20% sucrose gradient. - . - . acetetate activation; - propionate activation; - . . bovine serum albumin; - . ovalbumin. Journal of Dairy Science Vol. 64, No. 12, 1981

2340

RICKS AND COOK

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Figure 5. Effect of acetate concentration on the activity of acetyl CoA synthetase purified from heart mitochondria. The inset on the left is the Lineweaver-Burk plot, and the inset on the right is the Eadie-Hofstee plot of the same data.

from bovine or caprine mammary mitochondria (6, 15), although suitable for preparation of heart mitochondrial acetyl CoA synthetase, are not suitable for purification of the other acyl CoA synthetases, i.e., propionyl CoA synthetase and butyryt CoA synthetase of liver or kidney mitochondria (18, 19). With 10% glycerol during purification, a procedure which ensures that the unstable enzymes activating propionate and butyrate will be stabilized, and by monitoring all column effluents by acetate, propionate, and butyrate, we established that heart mitochondrial tissues contain high activity of one enzyme, acetyl CoA synthetase, with properties of activating short chain fatty acids. The enzyme activates primarily acetate and propionate (Figures 1 and 2, Table 1). Some evidence for an enzyme which activates butyrate was obtained (Table 1, Figure 1), and this probably is the same enzyme purified by Webster et al. (26). The physiological function of acetyl CoA Journal of Dairy Science Vol. 64, No. 12, 1981

synthetase in heart tissue would be to initiate oxidation of acetate obtained from peripheral blood to generate ATP needed for maintaining physiological function, i.e., pumping of blood. Heart tissue also can use medium and long chain fatty acids as sources of energy, and, thus, the physiological function of the butyrate

TABLE 2. Carbohydrate content of acetyl CoA synthetase purified from bovine heart mitochondria,a Monosaccharide

(pg/mg protein)

D-mannose D-galactose D-glucose N-acetyl-galactosamine N-acetyl-glucosamine

5.2 8.3 2.5 11.5 3.0

aSamples were analyzed by gas liquid chromatography of the trimethylsilyl derivatives of methyl glycosides.

BOVINE HEART ACYL CoA SYNTHETASES enzyme purified by Webster et al. (26) would possibly be the /3-oxidation of medium chain fatty acids for energy. It is unlikely that the enzyme binds significant quantities ofruminally derived butyrate, because butyrate mainly is metabolized by the rumen epithelial tissue to &hydroxybutyrate. Little butyrate appears in portal or peripheral blood. Thus, only the enzyme activating acetate is of significance in the animal's ability to use ruminally derived volatile fatty acids. Acetyl CoA synthetase was judged to be pure by the technique of polyacrylamide gel electrophoresis. Electrophoresis in the presence of SDS gave one band indicating that the enzyme probably was composed of a single polypeptide chain. The purified enzyme showed a tendency to bind to glass, a property that was not shared by the acyl CoA synthetases of liver (18). Moreover, acetyl CoA synthetase is stable relative to the other acyl CoA synthetases. Both binding and stability may be related to the enzyme being aglycoprotein (Table 2). Enzymes which are glycoproteins are relatively stable to degradation by proteolytic enzymes (5) and are remarkably stable on storage and at elevated temperatures (1, 16). In addition, enzymes composed of a single polypeptide chain are generally more stable than oligomeric enzymes (22). Compagnari and Webster (3) were unable to detect carbohydrate in their preparation of heart acetyl CoA synthetase. Acetyl CoA synthetase has been purified from bovine mammary gland mitochondria (14, 15), and this enzyme has many properties that are similar to those of the acetyl CoA synthetase isolated from heart tissue. Both are glycoproteins, readily aggregate, have similar apparent molecular weights, and behave similarly on chromatography on DEAE--23 cellulose and calcium phosphate gel. The affinity of the enzyme for acetate is similar. The K m is 6.1 × 10 - 4 M for the mammary acetyl CoA synthetase (15), and the K m for the heart acetyl CoA synthetase is 1.8 × 10--4M (Figure 5). The mammary enzyme has a similar pattern of substrate activation as the heart enzyme (Table 3) (14). Both enzymes have a high affinity for acrylate. The relative activity of heart acetyl CoA synthetase for acetate, propionate, and acrylate is 100:67:140 (Table 3), whereas the relative activity of the mammary gland enzyme

2 3 41

for these substrates is 100:65:141 (14). Both enzymes are active over a rather broad pH range, and both are inhibited weakly by AMP (17, 14). The enzymes are probably not identical. The mammary enzyme contains fructose, galaetose, glucose, and N-acetyl-neuraminic acid (22), whereas the heart enzyme contains mannose, galactose, glucose, N-acetyl-galactosamine, and N-acetyl-glucosamine but not fucose (Table 2). Activity of the enzymes in mammary tissue is dependent on stage of lactation (11), whereas the enzyme in heart tissue probably does not demonstrate large fluctuations in activity from changes in physiological state (17). Acetate concentration in peripheral blood of dairy cows is approximately 1 to 3 mM (17) and can increase slightly after feeding (20). Significant quantities of acetate are not removed from portal blood by liver as unlike nonruminant animals, acetyl CoA synthetase activity is low (18). We speculate that this adaptation has occurred because of the low absorption of dietary hexose in these animals. This ensures that an energy source such as acetate is available for continuing functioning of various body organs such as heart. In addition to greater availability of acetate in peripheral blood of ruminants relative to nonruminant animals (2), kinetic properties of heart acetyl CoA synthetase ensure continual uptake of acetate from blood. The K m for acetate is 1.79 X 10--4M and 6.1 × 10--4M for heart and mammary acetyl CoA synthetases.

TABLE 3. Substrate specificity of acetyl CoA synthetase purified from bovine heart mitochondria, a Substrates tested

Relative activity

Acetate Propionate Butyrate Valerate Hexanoate Heptanoate Octanoate Acrylate Maleate Crotonate

100 67 0 0 0 0 0 140 0 19

aFatty acids (racemic mixtures) were used at a concentration of 5/zmol. Journal of Dairy Science Vol. 64, No. 12, 1981

2342

RICKS AND COOK

Thus, the heart enzyme will be at half maximal velocity when acetate concentration is approximately. 179 mM. Acetate concentration in peripheral blood is far greater than the Km, and because acetate diffuses readily across cell membranes, acetate concentration per se probably does n o t control its rate of uptake by heart or lactating mammary gland mitochondria. The enzyme will be saturated with substrate at all physiological concentrations of acetate. Presumably both heart and lactating mammary tissue are critically dependent on a constant supply of acetate for energy. In the former case it is required for mechanical work, i.e., pumping blood, and in the latter case for synthesis and secretion of milk. Thus, kinetic properties of the enzyme ensure that as much acetate as is available will be taken up and that fluctuations in availability of substrate (acetate) will not influence enzyme activity. Uptake of acetate by heart tissue in the adult ruminant, therefore, is not controlled simply by substrate availability as has been demonstrated for the propionyl CoA synthetase of liver (18). At all physiological concentrations of blood acetate, acetyl CoA synthetase will be at maximal velocity. This does n o t preclude some other mechanism such as feedback inhibition (allosteric regulation) from controlling acetyl CoA synthetase activity in vivo. However, this seems unlikely because the enzyme is composed of a single polypeptide chain of molecular weight 67,500. In general, allosteric enzymes are composed of a number of polypeptide chains or subunits. Metabolic regulation of acetate uptake, therefore, must be governed by the amount of enzyme within a given tissue. Acetyl CoA synthetase activity in mammary tissue prior tO parturition is low, but activity increases as the gland becomes functional in milk synthesis and secretion (11). This increase is from an increase in enzyme synthesis rather than conversion of an inactive to an active form of the enzyme. Moreover, it appears to be under hormonal regulation. In the adult dairy cow, acetyl CoA synthetase activity of heart tissue does not show these fluctuations with lactational state (17), and this would be compatible with the idea that continual uptake of acetate, irrespective of physiological and nutritional state, would be required for efficient metabolic functioning of this organ.

Journal of Dairy Science Vol. 64, No. 12, 1981

Additional evidence in support of the idea that large fluctuations in acetyl CoA synthetase activity of heart tissue do not occur has been provided by an experiment in which enzyme activity of heart tissue in the fetus and young calf to 120 days of age has been measured (17). Enzyme activity was high in the fetus, at birth, and in older animals, irrespective of whether this latter group was fed a liquid (all milk) or solid (hay/grain) diet. Although these results conflict with those of Warshaw (25), who found a deficiency in acetyl CoA synthetase in bovine fetal heart tissue, they do suggest that acetyl CoA synthetase activity of heart mitochondria develops irrespective of whether the animal has a system of metabolism based on glucose (liquid diet fed group) or on glucose and fatty acids (normal fed group). Because of placental transfer, acetate is in fetal blood (17). It is, therefore, not surprising that fetal heart tissue activates acetate. Fetal heart tissue, unlike many other fetal organs, is functional early in gestation. REFERENCES

1 Arnold, W. N. 1969. Heat inactivation kinetics of yeast /3-fructofuranosidase. A polydisperse system. Biochim. Biophys. Acta 178:347. 2 Ballard, F. J. 1972. Supply and utilization of acetate in mammals. Amer. J. Clin. Nutr. 25:773. 3 Campagnari, F., and L. T. Webster, Jr. 1963. Purification and properties of acetyl coenzyme A synthetase from bovine heart mitochondria. J. Biol. Chem. 238:1628. 4 Chrambach, A., R. A. Reisfield, M. Wyckoff, and J. Zaccari. 1971. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Biochem. 20:150. 5 Coffey, J. W., and C. DeDuve. 1968. Digestive activity of lysosomes. I. The digestion of proteins by extracts of rat liver lysosomes. J. Biol. Chem. 243:3255. 6 Cook, R. M., S. Simon, and C. A. Ricks. 1975. Utilization of volatile fatty acids in ruminants. VI. Purification of acetyl coenzyme A synthetase from mitochondria of lactating goat mammary gland. J. Agric. Food Chem. 23:561. 7 Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. New York Acad. Sci. 121:404. 8 Hotchkiss, R. D. 1948. A microchemical reaction resulting in the staining of polysaccharide structure in fixed tissue preparations. Arch. Biochem. Biophys. 16:131. 9 Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265.

BOVINE HEART ACYL CoA SYNTHETASES 10 Mahler, H. R., S. J. Wakil, and R. M. Bock. 1953. Studies on fatty acid oxidation. I. Enzymatic activation of fatty acids. J. Biol. Chem. 204:453. 11 Marinez, D. I., C. A. Ricks, and R. M. Cook. 1976. Utilization of volatile fatty acids in ruminants. 8. Acetate activation in mammary tissue. J. Agric. Food Chem. 24:927. 12 Martin, R. G., and B. N. Ames. 1961. A method for determining the sedimentation behavior of enzymes: application to protein mixture. J. Biol. Chem. 236:1372. 13 Quraishi, S., and R. M. Cook. 1972. Utilization of volatile fatty acids in ruminants. IV. Relative activities of acetyl CoA synthetase and acetyl CoA hydrolase in mitochondria and intracellular localization of acetyl CoA synthetase. J. Agric. Food Chem. 20:91. 14 Qureshi, S. 1971. Studies on the isolation and purification of acetyl CoA synthetase from mitochondria of lactating bovine mammary gland. Ph.D. thesis, Michigan State Univ., East Lansing. 15 Qureshi, S., and R. M. Cook. 1975. Utilization of volatile fatty acids in ruminants. V. Purification of acetyl coenzyme A synthetase from mitochondria of lactating bovine mammary gland. J. Agric. Food Chem. 23:555. 16 Razur, J. H., H. R. Knull, and D. L. Simpson. 1970. Glycoenzymes: a note on the role for the carbohydrate moieties. Biochem. Biophys. Res. Comm. 40:110. 17 Ricks, C. A. 1979. Regulation of volatile fatty acid uptake by bovine heart, liver and kidney tissue.

2 34 3

Ph.D. thesis, Michigan State Univ., East Lansing. 18 Ricks, C. A., and R. M. Cook. 1981. Regulation of volatile fatty acid uptake by mitochondrial acyl CoA synthetases of bovine liver. J. Dairy Sci. 64:2324. 19 Ricks, C. A., and R. M. Cook. 1981. Partial purification of enzymes of bovine kidney mitochondria activating volatile fatty acids. J. Dairy Sci. 64: 2344. 20 Ross, J. P., and W. D. Kitts. 1973. Relationship between postprandial plasma volatile fatty acids, glucose and insulin levels in sheep fed different feeds. J. Nutr. 103:488. 21 Segal, I. M. 1976. In Biochemical calculations. 2nd ed. John Wiley and Sons, Inc., New York, NY. 22 Stamoudis, V. 1973. Acetyl CoA synthetase, a glycoprotein. M. S. thesis, Michigan State Univ., East Lansing. 23 Warburg, O., and W. Christian. 1941. Isolierung und kristallisation des garungsferments enolase. Biochem. Z. 310:384. 24 Warren, L. 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234:1971. 25 Warshaw, J. B. 1970. Cellular energy metabolism during fetal development. III. Deficient acetyl-CoA synthetase, acetyl carnitine transferase and oxidation of acetate in the fetal bovine heart. Biochim. Biophys. Acta 223:409. 26 Webster, L. T., Jr., L. D. Gerowin, and L. Rakita. 1965. Purification and characteristics of a butyryl coenzyme A synthetase from bovine heart mitochondria. J. Biol. Chem. 240:29.

Journal of Dairy Science Vol. 64, No. 12, 1981