An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.
I . Two young Friesian steers fitted with rumen cannulas were each given three different isonitrogenous and isoenergetic diets for successive periods of 2-3 weeks. The diets consisted mainly of straw and tapioca, with the nitrogen supplied mainly as decorticated groundnut meal (DCGM; diet A), in approximately equal amounts of DCGM and urea (diet B), or entirely as urea (diet C).2. At the end of each period on a given diet, part of the dietary urea of a morning feed was replaced by a solution of [lSN]urea which was infused into the rumen. Samples of rumen contents were removed just before giving the 15N dose and at I , 3 , s , 7 and 24 h afterwards, concentrations of ammonia and its I5N enrichment were determined and samples of mixed bacteria were prepared. Amino acids, ammonia derived mainly from amide groups, and hexosamines were prepared by ion-exchange chromatography of acid-hydrolysates of the bacteria and analysed for 15N.3. Approximate estimates of net bacterial N synthesis were made from turnover data for rumen fluid and I5N enrichments in rumen fractions. From the determined efficiency of incorporation of urea-N into bacteria recovered at the duodenum, it was calculated that on diets A, B and C respectively 82 x, 37 and o % of the bacterial N was derived from dietary protein or other non-urea sources.4. ['5N]urea was converted rapidly to ammonia and the 15N then incorporated into bacterial amide-N; it appeared at a slower rate in total bacterial non-amide-N. Rates of incorporation into non-amide-N were highest for glutamic acid, aspartic acid and alanine, and generally lowest for proline (pro), histidine (his), phenylalanine (phe), arginine (arg), methionine (met) and galactosamine. A similar ranking was also generally observed for relative 15N abundances (15N atoms "/, excess in N componentt15N atoms % excess in total bacterial N) achieved after several hours. Relative 15N abundances in his, arg and pro increased with decreasing protein @CGM) in the diet but those in the other protein amino acids, including the poorly labelled met, phe (and its derivative tyrosine) did not.5. It was concluded that different extents of labelling of the amino acids (at least those present mainly in protein) indicated that different amounts of preformed units (amino acids or peptides) were used. When an adequate supply of such units was available (particularly on diet A) pro, arg, his, met and phe were derived in this way to a greater extent than the other amino acids, but whereas synthesis of pro, arg and his increased on the low-protein diet C, that of met and phe did not. Thus met and phe may be limiting for bacterial growth on diets low in protein and high in non-protein-N.6. Differences in the extent of labelling of other bacteria1 N components may be due to different turnover rates.
The influence on protein accretion and whole-body protein turnover of changing dietary protein quality while maintaining constant energy intake was studied by varying the degree of lysine supplementation of a lysine-deficient barley-based diet given to growing pigs. Measurements of nitrogen metabolism and whole-body protein turnover, using both classical and 15N end-product methods following a single dose of [I5N]glycine, were made in 49-kg male pigs given diets containing 109 g lysine-deficient protein/kg supplemented to make them (1) 'deficient', (2) 'adequate' and (3) 'in excess' with respect to lysine. The "N dose and protein intake values used to calculate amino N flux from the cumulative urinary excretion of "N in urea and ammonia were corrected respectively for apparent digestibilities of (15N]glycine and total N determined in a separate experiment in pigs fitted with simple ileal cannulas. N retention and biological value were significantly increased by lysine supplementation of the deficient diet to the 'adequate' level, but were not further increased by the higher level of supplementation. Rates of growth paralleled these changes. The poorer biological value of the unsupplemented diet 1 was shown also in a significantly higher excretion of urea N compared with diets 2 and 3. N digestibility was not markedly influenced by the level of lysine supplementation. Both whole-body protein synthesis and degradation increased markedly on 'adequate' supplementation of the diet with lysine, but did not increase further with an excess of lysine. It is concluded that the increase in protein accretion rate observed on supplementation of the diet with lysine was due to a greater increase in the rate of protein synthesis than of degradation, rather than a decrease in degradation rate.Growth : Lysine requirement: Protein turnover: Pig
SummaryThe folate in cow's milk was strongly and specifically bound to a minor whey protein (FP), forming a complex of primary M of about 38000, but exhibiting concentration-dependent reversible aggregation. The binding protein was present in excess, and the milk had the capacity to bind about 50 μg added folic acid/l. An enriched concentrate of FP was prepared by ammonium sulphate fractionation—FP was precipitated at between 50 and 60% saturation—and further purified by chromatography in DEAE-cellulose and filtration in Sephadex gel G150. Its identity as a distinct minor whey protein was confirmed by comparative starch gel electrophoresis at various pH values.Some properties of the protein are described, and its physiological significance discussed.
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