Additional Methionine Research Articles

Protected Methionine and Heat-Treated Soybean Meal for High Producing Dairy Cows

Twenty-four high producing Holstein cows (14 primiparous and 10 multiparous) were fed concentrate mix containing heat-treated soybean meal without or with 15g of added DL-methionine daily provided as 50g of ruminally protected methionine product during wk 4 through 16 postpartum. The 15% crude protein mixed diets contained 30% (dry matter basis) corn silage, 15% alfalfa hay, and 55% concentrate mix. Covariant adjusted yields of milk (34.6 and 33.1 kg/d), 4% fat-corrected milk (28.4 and 27.6 kg/d), and solids-corrected milk (29.0 and 28.5 kg/d) were not increased by feeding supplemental methionine. Percentages of fat (2.81 and 2.92) and protein (2.88 and 2.92) were similar, whereas total solids (11.49 and 12.69) and solids-not-fat (8.68 and 8.77) were higher with supplemental methionine. Dry matter intakes (20.2 and 21.0 kg/d) were higher when cows were fed supplemental methionine. Ruminal pH, volatile fatty acids, and ammonia as well as blood serum urea and glucose were generally unaffected by methionine supplementation. Concentrations of methionine in arterial and venous plasma were elevated slightly when additional methionine was fed. The first-limiting amino acid for milk production, methionine, was calculated by several methods and was not changed by feeding supplemental ruminally protected methionine.

Human growth hormone produced with recombinant DNA technology: development and production.

The molecular basis of recombinant DNA technology is described, and the principles of genetically engineered proteins developed. The production of hGH by such methods utilizes a strain of Escherichia coli as host and a vector plasmid containing the appropriate information. Fermentation and purification of the hGH produced gives a preparation of high purity, containing only 1-2 ppm of E. coli polypeptide (ECP). This somatrem (Somatonorm) is identical to pituitary hGH except for an additional methionine residue at the N-terminal. Monoclonal antibodies fail to distinguish between pituitary hGH and somatrem. Preclinical studies of a variety of pharmacological and toxicological parameters indicate that the two hGH preparations have identical biological effects; no toxicological or mutagenic effects of somatrem have been detected.

Separation of recombinant human interleukin-2 and methionyl interleukin-2 produced in [formula omitted

Escherichia coli harboring the gene coding for human interleukin-2 (IL-2) produced methionyl IL-2 (Met-IL-2) having an additional methionine residue at the amino terminus as well as IL-2 starting with the amino terminal alanine. IL-2 and Met-IL-2 were copurified from a cell-free extract. It was difficult to separate these two molecular species from each other because of the similarities of their physico-chemical characteristics. We found that the isoelectric points of IL-2 and Met-IL-2 were slightly but significantly different and succeeded in separating these two molecular species by utilizing the difference of their isoelectric points. The isoelectric points of IL-2 and Met-IL-2 thus obtained were determined to be 7.7 and 7.5, respectively. The in vitro specific activities of these two species were the same and similar to that of natural human IL-2 derived from peripheral blood lymphocytes.

Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.

Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein.

Early Application of Conventional or Ahemeral Photoperiods in an Attempt to Improve Egg Size

Treatments were arranged as a 2 × 2 × 3 × 2 factorial representing 2 housing ages, 2 17% crude protein diets with and without added methionine and corn oil, 3 weight groups, and 2 light treatments 14L:10D and 14L:14D. Leghorn pullets were classified as heavy, medium, or light weight at either 15 or 19 weeks of age, and at those times, moved to individual laying cages maintained in rooms with either a 14L:10D or 14L:14D photoperiod. Birds were fed a conventional 17% CP corn-soybean diet or one containing additional methionine and corn oil. Each factorial treatment was represented by four replicate groups of seven consecutive individually caged birds. The trial was terminated at 71 weeks of age. Ahemeral lighting resulted in increased egg weight, body weight, and inferior shell quality, whereas egg production and feed intake were reduced (P<.01). Early transfer at 15 weeks resulted in loss of egg numbers (P<.01), while body weight at transfer was positively correlated with egg production, feed intake, and egg weight. Diet had no effect on production.In a second and third trial Leghorn pullets were reared on constant photoperiods of 8 or 14 hr. At 15 or 19 weeks of age, pullets were transferred to environments providing regular or ahemeral cycles as described for Trial 1. Again, pullets were classified according to weight and fed diets of low or high nutrient density. Both trials were terminated at 31 weeks of age. The effects of ahemeral lighting in Trial 2 on food intake, egg weight, egg production, and body weight were as described in Experiment 1, although in this situation eggshell quality was improved. Rearing birds on 14 vs. 8 hr photoperiods, in both trials, resulted in loss in egg numbers (P<.01), although egg weight was increased (P<.01). A final trial also involved rearing on photoperiods of 8 vs. 14 hr for weight-segregated pullets fed regular or high nutrient density diets. Adult photoperiod was a constant 14 or 17 hr, and again the trial was terminated at 31 weeks of age. Heavier pullets, at transfer time of 19 weeks, produced the greatest amount of eggs that were also larger (P<.01) while consuming more feed (P<.01). A rearing photoperiod of 14 vs. 8 hr reduced egg numbers (P<.01) while increasing egg size (P<.05) and body weight (P<.01).It is concluded that ahemeral lighting can be used to stimulate early egg size, although this is at the expense of egg numbers. While increasing the rearing photoperiod from 8 to 14 hr stimulates pullet growth, subsequent egg production is reduced, possibly due to the insufficient photostimulation at time of transfer.

BROILER CARCASS COMPOSITION AS AFFECTED BY AMINO ACID SUPPLEMENTATION

A corn-soybean diet containing 20% protein and a similar diet with glycine supplementation, to give a diet with a 24% protein equivalent, were supplemented with lysine and methionine. Weight gain, feed intake and carcass composition of male broilers were compared to those of similar birds fed a 24% protein corn-soybean diet. Weight gain and feed:gain ratio for the amino-acid-supplemented 20% protein diet were not significantly different from the 24% protein diet. The addition of glycine, while having little effect on performance, resulted in a reduction (P &lt; 0.05) in carcass fat. In a second experiment, increasing the level of dietary protein to 22% from 16% or supplementing the 16% diet with additional lysine and methionine failed to alter weight gain or feed:gain ratio during the 4- to 7-wk finishing period. However, total carcass protein was increased and total carcass fat was reduced with the higher protein diet. Although the amount of total edible meat was similar for all diets, the 22% protein diet resulted in meat containing a greater percentage of protein than the other dietary treatments. An interesting observation was the decrease in protein and increase in fat contents of breast meat when additional methionine was added to a 16% protein diet. Key words: Amino acid supplementation, broiler carcass composition, meat yields

Self-Selection by Laying Hens of Diets Differentiated by Texture and Color

Laying hens were offered a choice of diets separated by texture, e.g., mash and pellets. The mash contained the energy-protein portion of the diet, and the pellets contained mainly the calcium portion. Supplementing the mash with additional methionine and lysine resulted in a significant (P<.05) improvement in egg size over similar unsupplemented diets. Coloring the pellet portion of the diet with a green vegetable dye resulted in egg shell quality that was significantly poorer than similar uncolored diets. Whether this was due to overconsumption or underconsumption of the high calcium diet at various times of the day could not be determined.

Expression of synthetic human lysozyme gene in Escherichia coli.

A 418 base pair DNA duplex consisting of a gene for human lysozyme with a synthetic ribosome binding site of E. coli was assembled by stepwise ligation of 56 chemically synthesized oligodeoxyribonucleotides. The synthesized gene was expressed under the control of the lambda pRpL promoter and tfie gene product accumulated as several percent of the total cellular proteins in E. coli. The expressed product existed as insoluble and biologically inactive aggregates in the cells, but the biological activity was regenerated by solubilization and renaturation of the aggregated protein. The results of the N-terminal amino acid sequence analysis of the product showed the human lysozyme synthesized in E. coli has an additional methionine residue at the N-terminal of the mature human lysozyme due to the translational initiation codon of E. coli.

Primary structure of human preangiotensinogen deduced from the cloned cDNA sequence.

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.

Nutritional Evaluation of Four Varieties of Colored and One of White Beans (Phaseolus vulgaris) for Japanese Quail

Abstract The nutritional values of one variety of white and four varieties of colored beans were determined using a quail growth assay. The beans contained 7.2 to 7.5% moisture, 3.7 to 6.0% ash, 2.1 to 2.7% lipid, and 21.9 to 25.3% protein. The amino acid profile of the beans was also determined. Trypsin inhibitor activity was highest in the bean varieties of Small White and Black Turtle Soup (89.8 and 90.7 trypsin inhibitor activity/mg, respectively). The nutritional value of the Small White bean in a test diet containing 24% protein, without additional methionine, was superior to the colored beans. When .4% methionine was supplemented, this nutritional difference disappeared. The Small White bean elicited the poorest growth response when incorporated at a level of 70% in test diets containing 24% protein and .4% methionine. Air-dried residues of the Small White bean, extracted with either water or 80% methanol, were lethal to growing quail, adult quail, and growing rats. The addition of the freeze-dried extracts to an isolated soybean protein control diet improved the physical appearance of the quail without influencing their body weight.

Nutritional Evaluation of Mung Beans (Phaseolus aureus) for Young Broiler Chickens

The feeding value of mung beans (Phaseolus aureus) was measured in two trials with young broiler chickens. Raw or boiled mung beans replaced soybean meal at graded levels up to 20% (Trial 1) or 40% (Trial 2) in corn-based diets. Additional oil, methionine, and lysine were included in the mung bean diets. Twenty-one day weight gains and feed conversions of chickens fed mung bean diets were similar (P>.05) to those of chickens fed control diets. Mortality was low in all treatments. Boiling the mung beans did not enhance their nutritional value. Pancreas weights were similar in chicks from all dietary treatments. Metabolizable energy (ME) of raw mung beans measured by a rapid technique using adult cockerels was 12.7 MJ/kg (3035 kcal/kg) dry matter.

Peptide Mapping Reveals Considerable Sequence Homology among the Three Polypeptide Subunits of G1 Storage Protein from French Bean Seed

The major storage protein, G1 globulin, of bean (cv. Tendergreen) seeds was subjected to limited proteolysis with trypsin, chymotrypsin, papain, proteinase K, and protease V8 and to cleavage with cyanogen bromide and 2-(2-nitrophenylsulfanyl)-3-methyl-3'bromoindolenine. Mapping of peptides separated from each of the three G1 subunits by polyacrylamide gel electrophoresis revealed that many proteolytic cleavage sites were present at similar positions on the subunits. Evidence was adduced that the G1 subunits are homologous in amino acid sequence for about 61% of their length. The remaining region (possibly COOH-terminal) of the subunits appears to be heterologous, with the alpha subunit bearing an additional methionine residue.

Effect of additional protein or methionine and threonine on tyrosine catabolism in rats fed diets high in tyrosine.

The supplementation of additional protein or methionine and threonine to a high tyrosinc-low protein diet has previously been shown to prevent the tyrosine toxicity. To elucidate the mechanism, studies were performed on the effect of these supplements on the capacity to oxidize excessive tyrosine. Male weanling rats were ad libitum fed a 10% casein diet containing 5% tyrosine with and without extra casein or methionine plus threonine for 7 days, then animals were injected intraperitoneally with l-tyrosine-U-14C and the oxidation rate to 14CO2 determined in vivo at an interval of several hours throughout a 24-hour period. The addition of extra casein or methionine and threonine to the high tyrosine diet enhanced the ability of tyrosine oxidation, and decreased the radioactivities of the TCA-soluble fractions in plasma, liver and muscle. A high level of free tyrosine in blood and tissues was also lowered by the addition of these supplements. The diurnal chenges in free tyrosine concentration of various tiss...

Response of liver tyrosine-catabolizing enzymes in rats fed high tyrosine diets.

Weanling rats were fed a 10% casein containing 5% tyrosine (10C5T) diet supplemented either with 20% casein or with 0.66% methionine plus 0.90% threonine for 7 days ad libitum, and then the activities of liver tyrosine-catabolizing enzymes: tyrosine (Tyr) transaminase; p-hydroxyphenylpyruvate (pHPP) hydroxylase; and homogentisate oxidase; were determined sequentially at various times during 24-hr period. The mean activity of Tyr transaminase was elevated by feeding high tyrosine diets, except the methionine and threonine supplemented diet. The mean activity of pHPP hydroxylase was depressed by high tyrosine but was reversed by the addition of extra casein or methionine and threonine, and the ratio of Tyr transaminase and pHPP hydroxylase activities in rats consuming the 10C5T diet was remarkably elevated. The mean activity of homogentisate oxidase was somewhat depressed in rats fed the 10C5T diet, but was not affected by the additional casein or methionine and threonine supplements. In all dietary groups,...

Utilization of 35S from radioactive methionine or sulfate in the detoxification of cyanide by rats.

Weanling rats were fed a semipurified diet based on 10% casein and 1,500 ppm potassium cyanide. The diet was supplemented with varying levels of DL-methionine and/or potassium sulfate such that all diets were iso-sulfurous except for the basal which had no additional sulfur. The animals were fed for 2 weeks after which half of the rats in all groups except the basal were used in a radioactive study. Urine was collected and analyzed for thiocyanate. Animals which received labeled methionine were more efficient in producing labeled thiocyanate than those which received labeled sodium sulfate, although the latter did contribute to the detoxification of cyanide. Increases due to additional methionine were larger for weight gain, feed consumption, and urinary and plasma thiocyanate than with supplemental sulfate.

Synthesis in vitro of ribosomal protein S20 and its precursor

I have purified and characterized two products synthesized in vitro in a system for coupled transcription and translation programmed by DNA from a transducing bacteriophage carrying the gene for ribosomal protein S20. One of these polypeptides appears to be identical with authentic S20 by several criteria, including its electrophoretic and chromatographic mobilities, and its ability to bind to 16S RNA. The second polypeptide is less basic than S20, but exhibits all the structural and functional properties of a precursor to S20, including the presence of an additional methionine residue, apparently as N-formylmethionine. Moreover, it is converted, albeit slowly, to S20 in cell-free extracts. The persistence of the precursor form of S40 may be functionally significant as well.

Glutamine as a precursor to N-terminal pyrrolid-2-one-5-carboxylic acid in mouse immunoglobulin λ-type light chains. Amino acid-sequence variability at the N-terminal extra piece of λ-type light-chain precursors

The mRNA molecules coding for three mouse immunoglobulin lambda-type light (L) chains (MOPC-104E lambda(1), RPC-20 lambda(1), MOPC-315 lambda(2)) programme the cell-free synthesis of precursors larger than the mature proteins. Radioactive amino acid-sequence analyses of each of the three precursors labelled with [(3)H]alanine, [(3)H]serine, [(3)H]glutamine, [(3)H]glutamic acid and [(3)H]threonine showed that an extra piece, at least 18 residues long, is linked to the N-terminus of the mature L-chains. The N-terminal extra-peptide segment may be 19 residues long, since analyses of precursors labelled with [(35)S]methionine indicated an additional N-terminal methionine residue which was recovered in low yields. Presumably this is the initiator methionine, which is known to be short lived in eukaryotes. The mature forms of MOPC-104E, RPC-20 and MOPC-315 lambda L-chains are blocked at the N-termini by pyrrolid-2-one-5-carboxylic acid (pyroglutamic acid). Sequence analyses of precursors labelled with [(3)H]glutamine and [(3)H]glutamic acid showed incorporation only of glutamine in a position that matches with the position of pyrrolid-2-one-5-carboxylic acid in the mature forms of all three precursors, and incorporation of glutamic acid in other positions. The data showed the absence of glutamine-glutamic acid interconversion, since the radioactive peaks obtained from either (3)H-labelled amino acid were discrete, and free from cross-contamination. These results prove that glutamine is the precursor amino acid of pyrrolid-2-one-5-carboxylic acid at the N-termini of the mature MOPC-104E lambda(1), RPC-20 lambda(1) and MOPC-315 lambda(2) L-chains. Thus the formation of pyrrolid-2-one-5-carboxylic acid by cyclization of glutamine is a post-translational event which occurs after, or concomitant with, cleavage of the extra piece from the precursor to yield the mature L-chain. The variable (V) regions (110 amino acid residues) of mouse lambda L-chains are quite similar: when compared with that of MOPC-104E lambda(1) chain, the V-region of RPC-20 lambda(1) chain differs in one residue, and the V-region of MOPC-315 lambda(2) chain differs in 11 residues. The partial sequence data show that the N-terminal extra pieces of the two lambda(1) L-chain precursors have, so far, identical partial sequences; the extra piece of the lambda(2) L-chain precursor differs from these in at least three out of 19 positions.

Facile alkylation of methionine by benzyl bromide and demonstration of fumarase inactivation accompanied by alkylation of a methionine residue.

Benzyl bromide is a selective alkylator of sulfur nucleophiles including methionine and cysteine. Only the mercaptide ion is a more efficient nucleophile than is the sulfur ether of methionine. Alkylation rates relative to methionine are 200: less than or equal to 0.03: less than or equal to 0.03: less than or equal to 0.02 for GS-, histidine, tryptophan, and GSH, respectively. Alkylation of methionine by benzyl bromide is more than 50 times faster than alkylation by iodoacetate. Fumarase is readily inactivated by exposure to benzyl bromide at pH 6.6 to 6.8 accompanied by alkylation of close to 1 methionine residue/subunit. Fumarase fully inactivated by exposure to benzyl bromide shows no detected alkylation of amino acid residues other than methionine. The rate of inactivation of fumarase by benzyl bromide is decreased about 4-fold by the presence of excess substrates. Denaturation of fumarase in 6 M urea at pH 6.5 exposes additional methionine as well as cysteine residues to alkylation.

Partial amino-acid sequence of the precursor of an immunoglobulin light chain containing NH2-terminal pyroglutamic acid.

Analyses of amino-acid sequences of the total cell-free products programmed by the mRNA of MOPC-104E gamma light (L)-chain show that over 95% of the products have sequences of a distinct protein that correspond to the L-chain precursor. In this precursor an extra piece is coupled to the NH2-terminus of the mature L-chain. Analyses of products labeled with [3H]alanine, [3H]leucine, and [3H]proline demonstrate that the extra piece is composed of at least 18 residues. Analyses of [35S]methione-labeled product indicate that the extra piece may contain an additional NH2-terminal methionine, which is detected in about 10% of the molecules. Partial recovery of the NJ2-terminal methionine (alanine, leucine, and proline are recovered in yields close to theoretical, greater than 95%) suggests that it is the initiator methionine, which is known to be short lived in eukaryotes due to rapid hydrolysis. Thus, the extra piece seems to be 19 residues in length, and it contains one methionine at the NH2-terminus, three alanines at positions 2, 12, and 17, and five leucines at positions 6, 8, 10, 11, and 13. The close gathering of leucine residues, as well as their abundance (26%), suggest that the extra piece would be quite hydrophobic. Hydrophobicity seems to be a general property of the extra piece, since similar clusters of leucine were found in the precursors of 3 KL-chains (Burstein, Y. & Schechter, I. (1976) Biochem. J. 157, 145-151). The NH2-terminus of the mature MOPC-104E gamma L-chain is blocked by pyroglutamic acid. The fact that in the precursor a peptide segment precedes this NH2-terminus establishes that pyroglutamic acid is not the initiator residue for synthesis of the L-chain. Apparently, the pyroglutamic acid is formed by cyclization of glutamic acid or glutamine during cleavage of the extra piece to yield the mature L-chain.

Amino Acid Supplementation of Turkey Breeder Rations

Beltsville Small White turkey hens (60) were randomized into four groups of 15 hens each, weighed and placed in individual laying cages. Immediately after lighting to initiate production, they were placed on a practical type turkey breeder diet calculated to contain 18.26% protein, 2893 Cal. M.E./kg., 3.13% Ca, 0.80% available P, 1.04% arginine, 0.86% lysine, 0.50% methionine & cystine and 0.24% tryptophane. Amino acids were added to this diet as follows: (1) none; (2) 0.1% lysine; (3) 0.05% methionine and (4) 0.1% lysine & 0.05% methionine.Lysine (0.1%) had no effect on reproductive performance when added to the basal ration or to the ration containing additional methionine. Methionine addition (0.05%) improved production significantly (P ≤ .005), improved feed efficiency, and increased egg size.