L-Methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro

Naomi L Hepburn, Christopher H Knight, Colin J Wilde, Kay AK Hendry, Hugh Galbraith

Research output: Contribution to journalArticle

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Abstract

L-methionine is a sulphur-containing nutritionally essential amino acid. It has a number of important roles in epidermal and dermal tissues of the integument of animals. Failure of normal function of these tissues in the hoof (claw) is a cause of lameness in cattle. Little is known about quantitative relationships between post-absorptive concentrations of nutrients including sulphur-containing amino acids and uptake and utilization by epidermis and dermis of the bovine claw. These parameters were studied at the tissue level by use of,in established hi vitro claw explant system using tissue from cattle of beef or dairy origin and L-[(35)S]-labelled methionine as tracer. The results showed that uptake of L-methionine by freshly prepared solear explants in Dulbecco's Modified Eagle Medium/F-12 Nutrient Mix (DMEM/F12) (1:1) medium containing 1.0mmol L-methionine/litre was concentrative after 5-8 min, essentially linear for up to 10 min and became curvilinear thereafter. Maximum uptake and steady state conditions were obtained at approximately 30 min. Further measurements were made following 21 h incubation in culture medium. Under conditions of varying concentrations of L-methionine and measurement of uptake after 30 min, the presence of a saturable curve, that obeyed Michaelis-Menten kinetics, was demonstrated. Values of 3.61 mmol/litre and 5.84 mmol/kg intracellular water/30 min were obtained for K(m) and V(max) respectively. Uptake was not influenced by L-cysteine and L-cystine concentrations in the culture media.

Similar culture and incubation conditions were used in subsequent studies of DNA and protein synthesis. These showed that rates of incorporation of L-methionine into protein fractions and stimulation of DNA synthesis measured by methyl-thymidine incorporation were dependent on L-methionine concentrations in the medium. Maximal rates occurred at approximately 50 Lrnol/litre, which is in the normal physiological range, and at 1 % of maximum uptake capacity. Examination of histological sections by autoradiography showed localization of L-[(35)S]-labelled methionine in basal and suprabasal epidermal cells with limited retention in dermis. Measurement, by a range of histological, immunohistochemical, electrophoretic, western blotting and autoradiographic techniques, provided further evidence of L-methionine-dependent regulation of proliferation, differentiation and synthesis of proteins under physiological concentrations, by epidermal hornforming cells.

A key role for L-methionine is Suggested in the production of horn in bovine claw. The extrapolation of these in vitro data provides guidance for strategies to optimize methionine supply to Claw tissues in vivo. Such extrapolation Suggests the appropriateness of delivery of systemic concentrations of 50 mu mol L-methionine/litre to maximize proliferative and protein depositional activity in solear epidermis and dermis in vivo.
Original languageEnglish
Pages (from-to)103-115
Number of pages13
JournalJournal of Agricultural Science
Volume146
Issue number1
Early online date9 Aug 2007
DOIs
Publication statusPublished - Feb 2008

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Hoof and Claw
claws
Methionine
methionine
explants
protein synthesis
uptake mechanisms
cattle
Proteins
dermis
Dermis
epidermis (animal)
Epidermis
Culture Media
tissues
In Vitro Techniques
sulfur
culture media
Sulfur Amino Acids
Food

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L-Methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro. / Hepburn, Naomi L ; Knight, Christopher H ; Wilde, Colin J ; Hendry, Kay AK ; Galbraith, Hugh.

In: Journal of Agricultural Science, Vol. 146, No. 1, 02.2008, p. 103-115.

Research output: Contribution to journalArticle

Hepburn, Naomi L ; Knight, Christopher H ; Wilde, Colin J ; Hendry, Kay AK ; Galbraith, Hugh. / L-Methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro. In: Journal of Agricultural Science. 2008 ; Vol. 146, No. 1. pp. 103-115.
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N2 - L-methionine is a sulphur-containing nutritionally essential amino acid. It has a number of important roles in epidermal and dermal tissues of the integument of animals. Failure of normal function of these tissues in the hoof (claw) is a cause of lameness in cattle. Little is known about quantitative relationships between post-absorptive concentrations of nutrients including sulphur-containing amino acids and uptake and utilization by epidermis and dermis of the bovine claw. These parameters were studied at the tissue level by use of,in established hi vitro claw explant system using tissue from cattle of beef or dairy origin and L-[(35)S]-labelled methionine as tracer. The results showed that uptake of L-methionine by freshly prepared solear explants in Dulbecco's Modified Eagle Medium/F-12 Nutrient Mix (DMEM/F12) (1:1) medium containing 1.0mmol L-methionine/litre was concentrative after 5-8 min, essentially linear for up to 10 min and became curvilinear thereafter. Maximum uptake and steady state conditions were obtained at approximately 30 min. Further measurements were made following 21 h incubation in culture medium. Under conditions of varying concentrations of L-methionine and measurement of uptake after 30 min, the presence of a saturable curve, that obeyed Michaelis-Menten kinetics, was demonstrated. Values of 3.61 mmol/litre and 5.84 mmol/kg intracellular water/30 min were obtained for K(m) and V(max) respectively. Uptake was not influenced by L-cysteine and L-cystine concentrations in the culture media. Similar culture and incubation conditions were used in subsequent studies of DNA and protein synthesis. These showed that rates of incorporation of L-methionine into protein fractions and stimulation of DNA synthesis measured by methyl-thymidine incorporation were dependent on L-methionine concentrations in the medium. Maximal rates occurred at approximately 50 Lrnol/litre, which is in the normal physiological range, and at 1 % of maximum uptake capacity. Examination of histological sections by autoradiography showed localization of L-[(35)S]-labelled methionine in basal and suprabasal epidermal cells with limited retention in dermis. Measurement, by a range of histological, immunohistochemical, electrophoretic, western blotting and autoradiographic techniques, provided further evidence of L-methionine-dependent regulation of proliferation, differentiation and synthesis of proteins under physiological concentrations, by epidermal hornforming cells. A key role for L-methionine is Suggested in the production of horn in bovine claw. The extrapolation of these in vitro data provides guidance for strategies to optimize methionine supply to Claw tissues in vivo. Such extrapolation Suggests the appropriateness of delivery of systemic concentrations of 50 mu mol L-methionine/litre to maximize proliferative and protein depositional activity in solear epidermis and dermis in vivo.

AB - L-methionine is a sulphur-containing nutritionally essential amino acid. It has a number of important roles in epidermal and dermal tissues of the integument of animals. Failure of normal function of these tissues in the hoof (claw) is a cause of lameness in cattle. Little is known about quantitative relationships between post-absorptive concentrations of nutrients including sulphur-containing amino acids and uptake and utilization by epidermis and dermis of the bovine claw. These parameters were studied at the tissue level by use of,in established hi vitro claw explant system using tissue from cattle of beef or dairy origin and L-[(35)S]-labelled methionine as tracer. The results showed that uptake of L-methionine by freshly prepared solear explants in Dulbecco's Modified Eagle Medium/F-12 Nutrient Mix (DMEM/F12) (1:1) medium containing 1.0mmol L-methionine/litre was concentrative after 5-8 min, essentially linear for up to 10 min and became curvilinear thereafter. Maximum uptake and steady state conditions were obtained at approximately 30 min. Further measurements were made following 21 h incubation in culture medium. Under conditions of varying concentrations of L-methionine and measurement of uptake after 30 min, the presence of a saturable curve, that obeyed Michaelis-Menten kinetics, was demonstrated. Values of 3.61 mmol/litre and 5.84 mmol/kg intracellular water/30 min were obtained for K(m) and V(max) respectively. Uptake was not influenced by L-cysteine and L-cystine concentrations in the culture media. Similar culture and incubation conditions were used in subsequent studies of DNA and protein synthesis. These showed that rates of incorporation of L-methionine into protein fractions and stimulation of DNA synthesis measured by methyl-thymidine incorporation were dependent on L-methionine concentrations in the medium. Maximal rates occurred at approximately 50 Lrnol/litre, which is in the normal physiological range, and at 1 % of maximum uptake capacity. Examination of histological sections by autoradiography showed localization of L-[(35)S]-labelled methionine in basal and suprabasal epidermal cells with limited retention in dermis. Measurement, by a range of histological, immunohistochemical, electrophoretic, western blotting and autoradiographic techniques, provided further evidence of L-methionine-dependent regulation of proliferation, differentiation and synthesis of proteins under physiological concentrations, by epidermal hornforming cells. A key role for L-methionine is Suggested in the production of horn in bovine claw. The extrapolation of these in vitro data provides guidance for strategies to optimize methionine supply to Claw tissues in vivo. Such extrapolation Suggests the appropriateness of delivery of systemic concentrations of 50 mu mol L-methionine/litre to maximize proliferative and protein depositional activity in solear epidermis and dermis in vivo.

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