Journal of Dairy Research (1979), 46, 69-73
Effects of restriction of amino acid supply to the isolated perfused guinea-pig mammary gland BY T. BEN MEPHAM, ANDREW R. PETERS AND STEPHEN ALEXANDROV* University of Nottingham, Department of Physiology and Environmental Studies, Faculty of Agricultural Science, Sutton Bonington, Loughborough, LE12 5RD (Received 27 June 1978) When individual essential amino acids were omitted for periods of 40-100 min from the infusate substrate solution in isolated perfused guinea-pig mammary gland experiments, uptake of methionine, tyrosine, phenylalanine, histidine and tryptophan (group 1) was significantly depressed by a mean of 49-8%, whereas the remaining essential amino acids (group 2) showed no significant decrease in uptake. During depletion periods oxidation of [14C]amino acids was increased. The possible significance of the differences in absorption between the 2 groups of amino acids is discussed. SUMMARY.
Previous work in this laboratory has shown that the isolated perfused guinea-pig mammary gland absorbs essential amino acids from the perfusate in amounts related to the output of these amino acids in milk protein (Davis & Mepham, 1974). We have suggested that essential amino acids belong to one of two categories, group 1, consisting of methionine, tyrosine, phenylalanine, histidine and tryptophan, the uptakes of which agree very closely with their outputs in milk protein, and group 2, consisting of threonine, valine, isoleucine, leucine, lysine and arginine, which are absorbed in excess of their requirement for protein synthesis (Davis & Mepham, 1976; Mepham, Peters & Davis, 19766; Peters, Alexandrov & Mepham, 1979). It has been demonstrated in several species that the group 2 amino acids are catabolized extensively in the mammary gland, e.g. arginine (Mepham, 1971), valine, isoleucine and leucine (Derrig, Davis & Clark, 1973; Davis & Mepham, 1976; Wohlt et al. 1977) and threonine (Verbeke et al. 1972). In contrast, the group 1 amino acids are catabolized to a lesser, and often negligible, extent, e.g. methionine (Verbeke, Simeonov & Peeters, 1967), tryptophan (Mepham et al. 19766), histidine and phenylalanine (Davis & Mepham, 1976). In experiments with the isolated perfused guinea-pig mammary gland, when the perfusate was depleted of tyrosine there was a marked difference in response of methionine, phenylalanine and histidine on the one hand and leucine on the other (Davis & Mepham, 1976). In the experiments reported here we have further examined the effect of depleting the perfusate or individual essential amino acids on the uptake and metabolism of other amino acids by the gland, in order to assess the importance of the 2 groups in controlling amino acid uptake and hence protein synthesis. Some of these results have been briefly reported in Bulgarian by Alexandrov, Peters & Mepham (1977). * Present address: Institute of Animal Breeding, Sofia, Bulgaria. 0022-0299/79/1685-1014S01.00 © 1979 Proprietors of the Journal of Dairy Eesearoh
70
T. B. MEPHAJM, A. R. PETERS AND S. ALEXANDROV
Table 1. Details of depletion experiments Expt
Duration,
no.
min
1 2 3 4 5 S 7 8 9 10
190 185 250 190 180 180 175 150 210 210
Deficient amino acid Methionine Methionine Leucine Leucine Histidine Histidine Arginine Tryptophan Valine Valine
Depletion period, min* 70-130 85-145 90-190 110-150 60-140 40-140 75-115 70-130 50-130 70-130
* The depletion period is the period during which the deficient amino acid was undetectable in the perfusate samples, except experiments 9 and 10 in which valine concentration was at 3/ig/ml (see text). The times shown are not all integrals of 20 min, because a varying equilibration period was allowed at the beginning of each perfusion before the first perfusate sample was taken. EXPERIMENTAL
Perfusion technique. The technique has been fully described by Davis & Mepham (1974) and Mepham, Davies & Humphreys (1976a). Experimental design. Experimental details are shown in Table 1. Ten perfusion experiments were performed in which a substrate solution deficient in one essential amino acid was infused from the beginning, thus resulting in the eventual absence of that amino acid from the perfusate (see Table 1). After a period of between 40 and 100 min (Table 1) the substrate solution was exchanged for one with the full complement of essential amino acids. Perfusate samples were taken at 20 min intervals for estimation of amino acid uptake. The effect of the lack of one essential amino acid on amino acid uptake and oxidation was studied. Amino acid analysis. This was carried out as described by Davis & Mepham (1976) except for tryptophan which was assayed using the method of Denckla & Dewey (1967) as modified by Lehmann (1971). Amino acid uptake was calculated using the serial sampling method described by Mepham et al. (1976ft). Amino acid oxidation. Radiochemicals were purchased from the Radiochemical Centre, Amersham, Bucks. Oxidation of amino acids was measured by trapping 14 CO2 in N-NaOH solution as described by Davis & Mepham (1976). Samples (1 ml) were taken from the NaOH reservoir at 15-min intervals and rates of accumulation of radioactivity calculated as d.p.m./min.
RESULTS AND DISCUSSION
Amino acid uptake. The perfusate was depleted of the appropriate amino acid (below the sensitivity of the amino acid analyser) in all cases except experiments 9 and 10, in which the concentration of valine fell to about 3/ig/ml, i.e. about 20% of the physiological concentration (Davis & Mepham, 1974). However, the response in these experiments was similar to that in the others (Peters, 1977). The effects of deficiency of an essential amino acid on uptake are shown in Table 2. In compiling this table the mean data for all experiments of the mean uptakes during each experiment were calculated. There were significant decreases in uptake of all group 1
Restriction of mammary amino acid supply
71
Table 2. Effect of essential amino acid depletion on amino acid uptake Amino acid Thr Val He Leu Lys Arg Met Tyr Phe His Trp Asp Ser Glu Gin Pro Gly Ala Orn
Mean uptake before and after depletion, mg/h (A) 1-42 + 0-1 206 + 0-23 1-62 ±008 3-00 + 0-18 2-66±0-17 1-75 + 009 0-89 ±004 113 + 006 0-98 ±0-09 0-72 ± 0 0 5 0-90±0-ll 004 + 005 0-83±0-18 4-51 ±0-51 1-50 ± 0 0 8 008 + 0-32 -0-80 ±0-25 -0-28 + 0-31 0-57 ±0-05
Mean uptake during depletion, mg/h (B)
1-29 ±008 l-37±014
1-34 + 0-11 2-45 + 0-20 2-34 ± 0 1 7 1-63 + 0-11 0-44 + 0-07 0-69 + 0-05 0-51 ±009 0-36 ±007 0-35 ±008 - 0 0 6 ±0-04 0-58 ±008 3-55 + 0-43 0-69 + 0-21 -0-21 ±0-20 -0-95 + 0-30 -12-2 + 0-25 0-59 + 0-03
Significance of difference (A-B» NS * NS NS NS NS *** *** ** *** ** NS
Decrease in uptake %
9-2x 33-5 1 7-3 1
\1CO_1_OO
16-2 ±3-9 121 6-9' 50-6, 390 480U9-8±3-5 500 61-2J 183
—
NS NS NS NS NS * —
332 —
Significance levels: NS, P > 0 0 5 ; * P < 0 0 5 ; ** P < 0 0 1 ; *** P < 0001. •f Using Student's t test.
Table 3. Oxidation data for depletion experiments Expt no. 1
2 3 4 5
6 8 10
Isotope* Valine Threonine Isoleucine Mothionine Leucine Arginine Isoleucine Lysine
Quantity, liCi 2x5 5
Time of injection, min
10, 110
5 5
25 25 30
2x5
40, 120
5 5
20 40 45
5
Oxidation rate before depletionf 2031 NV 665
2865 50 759
2538 2527
Oxidation rate during depletionf 3472 NV 910
111,480
Oxidation ratio % 1-71 NV 1-37 38-90
307
616
5015 3021 5432
6-60 1-19 2-15
NV, No values available. * All compounds used were L-[U- 1 4 C] labelled except for expt 4 in which L-[methyl-14C]methionine was used. f Oxidation rate expressed as d.p.m. per min per uptake (mg/h). % Rate during depletion/rate before depletion.
essential amino acids, while only valine of group 2 was significantly affected. The effect was similar irrespective of the deficient amino acid. The difference between the responses of groups 1 and 2 was significant in all experiments except in experiment 10. There was a highly significant difference (P < 0-001; Student's t test) between the overall percentage decrease in uptake of group 1 and that of group 2, the values being 49-8 ± 3-5 (S.E.M.) % and 16-2 + 3-9 % respectively. The uptakes of most of the non-essential amino acids were reduced during depletion, but the responses were very variable (Table 2). Alanine uptake was significantly reduced during depletion (P < 0-05), and since it was negative even before depletion this indicates an increased rate of synthesis by the gland. Ornithine
72
T. B . M E P H A M , A. R . P E T E R S AND S. A L E X A N D R O V
was the least affected non-essential amino acid. Resupplementation of the deficient amino acid stimulated a rapid return to normal absorption in all cases. Amino acid oxidation during depletion. The rate of oxidation is expressed as d.p.m. per min per unit uptake (mg/h) to compensate for any reduced uptake during depletion (Table 3). In all cases there was an increased rate of oxidation during depletion periods, indicating an increase in catabolism of amino acids generally. The pattern of oxidation in control experiments over equivalent time periods was invariably one of a decreased rate of oxidation following an exponential curve (Peters, 1977). In the present experiments, a group 1 amino acid isotope was used in only one experiment (Table 3), but the increase in oxidation during depletion was much higher than those of group 2, i.e. nearly 40-fold as compared to a factor of 3-2+ 1-02 for group 2 amino acids. However, due to the normally low degree of oxidation of methionine, this increase still did not represent a significant proportion of the methionine absorbed by the gland. Thus, the gland still had a considerable capacity for absorption of group 2 amino acids during depletion, as their absorption continued at about 85 % of the normal rate. Pep tide formation would presumably have continued for some time following depletion while the intracellular pool of the depleted amino acid was being reduced. There was, however, also an increase in the rate of amino acid oxidation and of the synthesis of some non-essential amino acids. For example, in experiment 6, 70% more radioactive label from [U-14C]arginine was recovered in proline and in experiment 2, 65% more label from [U-14C]threonine was recovered in glycine, than in control experiments utilizing the same isotopes (Peters, 1977). It is also possible that quite apart from metabolic interconversion the gland accumulated these amino acids, although analysis of tissue-free amino acids in perfused glands at the end of the experiments revealed no significant differences from those in glands from control experiments (Peters, 1977). However, in each experiment the deficient amino acid was re-infused (Table 1) so that tissue free amino acids may have returned to more normal levels by this time. The uptake of group 2 amino acids was apparently relatively independent of the rate of group 1 uptake and hence of the rate of protein synthesis, and of the availability and uptake of the other members of group 2. Davis & Mepham (1976) reported results of a perfusion experiment in which tyrosine was omitted from the perfusate in order to determine whether under such conditions synthesis of tyrosine from [14C]phenylalanine would occur. No significant transfer of 14C activity was detected, but while group 1 amino acid uptake was markedly depressed that of group 2 was little affected (although the designations 'group 1' and 'group 2' were not then used). The results reported in this paper show that depletion of the perfusate of any essential amino acid produces the same distinctive effect on the uptake of other essential amino acids. The increased oxidation of amino acids during periods when protein synthesis was depressed suggests that under these conditions oxidation merely serves to dispose of amino acids accumulated in excess of requirements. However, the remarkable pertinacity of the cells in continuing to absorb group 2 amino acids during depletion periods would seem to be of considerable, although as yet undefined, significance in relation to factors controlling milk protein synthesis.
Restriction of mammary amino acid supply
73
We are grateful to Mrs K. J. Lock for expert technical assistance. The work was supported by a research grant from the Agricultural Research Council. S. Alexandrov was in receipt of an International Atomic Agency Fellowship. REFERENCES ALEXANDROV, S., PETERS, A. R. & MEPHAM, T. B. (1977). Zhivotnovudni Nauki 14, 115.
DAVIS, S. R. & MEPHAM, T. B. (1974). Quarterly Journal of Experimental Physiology 59, 113. DAVIS, S. R. & MEPHAM, T. B. (1976). Biochemical Journal 156, 553. DENCKLA, W. D. & DEWEY, H. K. (1967). Journal of Laboratory and Clinical Medicine 69, 160. DERRIG, R. G., DAVIS, C. L. & CLARK, J. H. (1973). Journal of Dairy Science 56, 651.
LEHMANN, J. (1971). Scandinavian Journal of Clinical and Laboratory Investigation 28, 49. MEPHAM, T. B. (1971). In Lactation, p. 297. (Ed. I. R. Falconer.) London: Butterworths. MEPHAM, T. B., DAVIS, S. R. & HTTMPHREYS, J. R. (1976a). Journal of Dairy Research 43, 197. MEPHAM, T. B., PETERS, A. R. & DAVIS, S. R. (19766). Biochemical Journal 158, 659.
PETERS, A. R. (1977). Thesis, University of Nottingham. PETERS, A. R., ALEXANDROV, S. & MEPHAM, T. B. (1979). Journal of Dairy Research 46, 59. VERBEKE, R., ROETS, E., MASSART-LEEN, A.-M. & PEETERS, G. (1972). Journal of Dairy Research 39,
239. VERBEKE, R., SIMEONOV, S. & PEETERS, G. (1967). Archives Internationales de Physiologie et de Biochimie 75, 378. WOHLT, J. E., CLARK, J. H., DERRIG, R. G. & DAVIS, C. L. (1977). Journal of Dairy Science 60,1875.
Printed in Great Britain
Effects of restriction of amino acid supply to the isolated perfused guinea-pig mammary gland BY T. BEN MEPHAM, ANDREW R. PETERS AND STEPHEN ALEXANDROV* University of Nottingham, Department of Physiology and Environmental Studies, Faculty of Agricultural Science, Sutton Bonington, Loughborough, LE12 5RD (Received 27 June 1978) When individual essential amino acids were omitted for periods of 40-100 min from the infusate substrate solution in isolated perfused guinea-pig mammary gland experiments, uptake of methionine, tyrosine, phenylalanine, histidine and tryptophan (group 1) was significantly depressed by a mean of 49-8%, whereas the remaining essential amino acids (group 2) showed no significant decrease in uptake. During depletion periods oxidation of [14C]amino acids was increased. The possible significance of the differences in absorption between the 2 groups of amino acids is discussed. SUMMARY.
Previous work in this laboratory has shown that the isolated perfused guinea-pig mammary gland absorbs essential amino acids from the perfusate in amounts related to the output of these amino acids in milk protein (Davis & Mepham, 1974). We have suggested that essential amino acids belong to one of two categories, group 1, consisting of methionine, tyrosine, phenylalanine, histidine and tryptophan, the uptakes of which agree very closely with their outputs in milk protein, and group 2, consisting of threonine, valine, isoleucine, leucine, lysine and arginine, which are absorbed in excess of their requirement for protein synthesis (Davis & Mepham, 1976; Mepham, Peters & Davis, 19766; Peters, Alexandrov & Mepham, 1979). It has been demonstrated in several species that the group 2 amino acids are catabolized extensively in the mammary gland, e.g. arginine (Mepham, 1971), valine, isoleucine and leucine (Derrig, Davis & Clark, 1973; Davis & Mepham, 1976; Wohlt et al. 1977) and threonine (Verbeke et al. 1972). In contrast, the group 1 amino acids are catabolized to a lesser, and often negligible, extent, e.g. methionine (Verbeke, Simeonov & Peeters, 1967), tryptophan (Mepham et al. 19766), histidine and phenylalanine (Davis & Mepham, 1976). In experiments with the isolated perfused guinea-pig mammary gland, when the perfusate was depleted of tyrosine there was a marked difference in response of methionine, phenylalanine and histidine on the one hand and leucine on the other (Davis & Mepham, 1976). In the experiments reported here we have further examined the effect of depleting the perfusate or individual essential amino acids on the uptake and metabolism of other amino acids by the gland, in order to assess the importance of the 2 groups in controlling amino acid uptake and hence protein synthesis. Some of these results have been briefly reported in Bulgarian by Alexandrov, Peters & Mepham (1977). * Present address: Institute of Animal Breeding, Sofia, Bulgaria. 0022-0299/79/1685-1014S01.00 © 1979 Proprietors of the Journal of Dairy Eesearoh
70
T. B. MEPHAJM, A. R. PETERS AND S. ALEXANDROV
Table 1. Details of depletion experiments Expt
Duration,
no.
min
1 2 3 4 5 S 7 8 9 10
190 185 250 190 180 180 175 150 210 210
Deficient amino acid Methionine Methionine Leucine Leucine Histidine Histidine Arginine Tryptophan Valine Valine
Depletion period, min* 70-130 85-145 90-190 110-150 60-140 40-140 75-115 70-130 50-130 70-130
* The depletion period is the period during which the deficient amino acid was undetectable in the perfusate samples, except experiments 9 and 10 in which valine concentration was at 3/ig/ml (see text). The times shown are not all integrals of 20 min, because a varying equilibration period was allowed at the beginning of each perfusion before the first perfusate sample was taken. EXPERIMENTAL
Perfusion technique. The technique has been fully described by Davis & Mepham (1974) and Mepham, Davies & Humphreys (1976a). Experimental design. Experimental details are shown in Table 1. Ten perfusion experiments were performed in which a substrate solution deficient in one essential amino acid was infused from the beginning, thus resulting in the eventual absence of that amino acid from the perfusate (see Table 1). After a period of between 40 and 100 min (Table 1) the substrate solution was exchanged for one with the full complement of essential amino acids. Perfusate samples were taken at 20 min intervals for estimation of amino acid uptake. The effect of the lack of one essential amino acid on amino acid uptake and oxidation was studied. Amino acid analysis. This was carried out as described by Davis & Mepham (1976) except for tryptophan which was assayed using the method of Denckla & Dewey (1967) as modified by Lehmann (1971). Amino acid uptake was calculated using the serial sampling method described by Mepham et al. (1976ft). Amino acid oxidation. Radiochemicals were purchased from the Radiochemical Centre, Amersham, Bucks. Oxidation of amino acids was measured by trapping 14 CO2 in N-NaOH solution as described by Davis & Mepham (1976). Samples (1 ml) were taken from the NaOH reservoir at 15-min intervals and rates of accumulation of radioactivity calculated as d.p.m./min.
RESULTS AND DISCUSSION
Amino acid uptake. The perfusate was depleted of the appropriate amino acid (below the sensitivity of the amino acid analyser) in all cases except experiments 9 and 10, in which the concentration of valine fell to about 3/ig/ml, i.e. about 20% of the physiological concentration (Davis & Mepham, 1974). However, the response in these experiments was similar to that in the others (Peters, 1977). The effects of deficiency of an essential amino acid on uptake are shown in Table 2. In compiling this table the mean data for all experiments of the mean uptakes during each experiment were calculated. There were significant decreases in uptake of all group 1
Restriction of mammary amino acid supply
71
Table 2. Effect of essential amino acid depletion on amino acid uptake Amino acid Thr Val He Leu Lys Arg Met Tyr Phe His Trp Asp Ser Glu Gin Pro Gly Ala Orn
Mean uptake before and after depletion, mg/h (A) 1-42 + 0-1 206 + 0-23 1-62 ±008 3-00 + 0-18 2-66±0-17 1-75 + 009 0-89 ±004 113 + 006 0-98 ±0-09 0-72 ± 0 0 5 0-90±0-ll 004 + 005 0-83±0-18 4-51 ±0-51 1-50 ± 0 0 8 008 + 0-32 -0-80 ±0-25 -0-28 + 0-31 0-57 ±0-05
Mean uptake during depletion, mg/h (B)
1-29 ±008 l-37±014
1-34 + 0-11 2-45 + 0-20 2-34 ± 0 1 7 1-63 + 0-11 0-44 + 0-07 0-69 + 0-05 0-51 ±009 0-36 ±007 0-35 ±008 - 0 0 6 ±0-04 0-58 ±008 3-55 + 0-43 0-69 + 0-21 -0-21 ±0-20 -0-95 + 0-30 -12-2 + 0-25 0-59 + 0-03
Significance of difference (A-B» NS * NS NS NS NS *** *** ** *** ** NS
Decrease in uptake %
9-2x 33-5 1 7-3 1
\1CO_1_OO
16-2 ±3-9 121 6-9' 50-6, 390 480U9-8±3-5 500 61-2J 183
—
NS NS NS NS NS * —
332 —
Significance levels: NS, P > 0 0 5 ; * P < 0 0 5 ; ** P < 0 0 1 ; *** P < 0001. •f Using Student's t test.
Table 3. Oxidation data for depletion experiments Expt no. 1
2 3 4 5
6 8 10
Isotope* Valine Threonine Isoleucine Mothionine Leucine Arginine Isoleucine Lysine
Quantity, liCi 2x5 5
Time of injection, min
10, 110
5 5
25 25 30
2x5
40, 120
5 5
20 40 45
5
Oxidation rate before depletionf 2031 NV 665
2865 50 759
2538 2527
Oxidation rate during depletionf 3472 NV 910
111,480
Oxidation ratio % 1-71 NV 1-37 38-90
307
616
5015 3021 5432
6-60 1-19 2-15
NV, No values available. * All compounds used were L-[U- 1 4 C] labelled except for expt 4 in which L-[methyl-14C]methionine was used. f Oxidation rate expressed as d.p.m. per min per uptake (mg/h). % Rate during depletion/rate before depletion.
essential amino acids, while only valine of group 2 was significantly affected. The effect was similar irrespective of the deficient amino acid. The difference between the responses of groups 1 and 2 was significant in all experiments except in experiment 10. There was a highly significant difference (P < 0-001; Student's t test) between the overall percentage decrease in uptake of group 1 and that of group 2, the values being 49-8 ± 3-5 (S.E.M.) % and 16-2 + 3-9 % respectively. The uptakes of most of the non-essential amino acids were reduced during depletion, but the responses were very variable (Table 2). Alanine uptake was significantly reduced during depletion (P < 0-05), and since it was negative even before depletion this indicates an increased rate of synthesis by the gland. Ornithine
72
T. B . M E P H A M , A. R . P E T E R S AND S. A L E X A N D R O V
was the least affected non-essential amino acid. Resupplementation of the deficient amino acid stimulated a rapid return to normal absorption in all cases. Amino acid oxidation during depletion. The rate of oxidation is expressed as d.p.m. per min per unit uptake (mg/h) to compensate for any reduced uptake during depletion (Table 3). In all cases there was an increased rate of oxidation during depletion periods, indicating an increase in catabolism of amino acids generally. The pattern of oxidation in control experiments over equivalent time periods was invariably one of a decreased rate of oxidation following an exponential curve (Peters, 1977). In the present experiments, a group 1 amino acid isotope was used in only one experiment (Table 3), but the increase in oxidation during depletion was much higher than those of group 2, i.e. nearly 40-fold as compared to a factor of 3-2+ 1-02 for group 2 amino acids. However, due to the normally low degree of oxidation of methionine, this increase still did not represent a significant proportion of the methionine absorbed by the gland. Thus, the gland still had a considerable capacity for absorption of group 2 amino acids during depletion, as their absorption continued at about 85 % of the normal rate. Pep tide formation would presumably have continued for some time following depletion while the intracellular pool of the depleted amino acid was being reduced. There was, however, also an increase in the rate of amino acid oxidation and of the synthesis of some non-essential amino acids. For example, in experiment 6, 70% more radioactive label from [U-14C]arginine was recovered in proline and in experiment 2, 65% more label from [U-14C]threonine was recovered in glycine, than in control experiments utilizing the same isotopes (Peters, 1977). It is also possible that quite apart from metabolic interconversion the gland accumulated these amino acids, although analysis of tissue-free amino acids in perfused glands at the end of the experiments revealed no significant differences from those in glands from control experiments (Peters, 1977). However, in each experiment the deficient amino acid was re-infused (Table 1) so that tissue free amino acids may have returned to more normal levels by this time. The uptake of group 2 amino acids was apparently relatively independent of the rate of group 1 uptake and hence of the rate of protein synthesis, and of the availability and uptake of the other members of group 2. Davis & Mepham (1976) reported results of a perfusion experiment in which tyrosine was omitted from the perfusate in order to determine whether under such conditions synthesis of tyrosine from [14C]phenylalanine would occur. No significant transfer of 14C activity was detected, but while group 1 amino acid uptake was markedly depressed that of group 2 was little affected (although the designations 'group 1' and 'group 2' were not then used). The results reported in this paper show that depletion of the perfusate of any essential amino acid produces the same distinctive effect on the uptake of other essential amino acids. The increased oxidation of amino acids during periods when protein synthesis was depressed suggests that under these conditions oxidation merely serves to dispose of amino acids accumulated in excess of requirements. However, the remarkable pertinacity of the cells in continuing to absorb group 2 amino acids during depletion periods would seem to be of considerable, although as yet undefined, significance in relation to factors controlling milk protein synthesis.
Restriction of mammary amino acid supply
73
We are grateful to Mrs K. J. Lock for expert technical assistance. The work was supported by a research grant from the Agricultural Research Council. S. Alexandrov was in receipt of an International Atomic Agency Fellowship. REFERENCES ALEXANDROV, S., PETERS, A. R. & MEPHAM, T. B. (1977). Zhivotnovudni Nauki 14, 115.
DAVIS, S. R. & MEPHAM, T. B. (1974). Quarterly Journal of Experimental Physiology 59, 113. DAVIS, S. R. & MEPHAM, T. B. (1976). Biochemical Journal 156, 553. DENCKLA, W. D. & DEWEY, H. K. (1967). Journal of Laboratory and Clinical Medicine 69, 160. DERRIG, R. G., DAVIS, C. L. & CLARK, J. H. (1973). Journal of Dairy Science 56, 651.
LEHMANN, J. (1971). Scandinavian Journal of Clinical and Laboratory Investigation 28, 49. MEPHAM, T. B. (1971). In Lactation, p. 297. (Ed. I. R. Falconer.) London: Butterworths. MEPHAM, T. B., DAVIS, S. R. & HTTMPHREYS, J. R. (1976a). Journal of Dairy Research 43, 197. MEPHAM, T. B., PETERS, A. R. & DAVIS, S. R. (19766). Biochemical Journal 158, 659.
PETERS, A. R. (1977). Thesis, University of Nottingham. PETERS, A. R., ALEXANDROV, S. & MEPHAM, T. B. (1979). Journal of Dairy Research 46, 59. VERBEKE, R., ROETS, E., MASSART-LEEN, A.-M. & PEETERS, G. (1972). Journal of Dairy Research 39,
239. VERBEKE, R., SIMEONOV, S. & PEETERS, G. (1967). Archives Internationales de Physiologie et de Biochimie 75, 378. WOHLT, J. E., CLARK, J. H., DERRIG, R. G. & DAVIS, C. L. (1977). Journal of Dairy Science 60,1875.
Printed in Great Britain
