Isolated membranes of Pseudomonas aeruginosa were found to bind radioactive isoleucine and proline, two amino acids for which active transport systems are known. The active transport systems in whole cells of this organism are energy dependent; the binding systems in isolated membrane preparations are not energy dependent, but are inducible, require magnesium ions and are stable to short periods of sonication usually sufficient to destroy whole cells. An assay measuring the binding of radioactive amino acid to amino acid binding protein present in isolated membrane preparations of P. aeruginosa was developed and discussed. Cells induced to high levels of amino acid transport produced equivalent levels of binding on isolation of membranes from these cells. Cells repressed for amino acid transport did not lose a corresponding level of binding on isolation of their membranes, suggesting involvement of more than one protein in the active transport system of that particular amino acid. Evidence was found to substantiate claims that active transport systems are family specific, however it was also determined that the aliphatic amino acid binding system was not stereospecific. Isolated membrane preparations of P. aeruginosa were found to produce adenosine triphosphate, but this energy-rich, phosphate bond compound did not appear to function in binding of radioactive amino acid to membrane preparations. Its possible functions are discussed. Several methods of isolation of proteins with binding properties from isolated membranes and from osmotic shock supernatant fluids were attempted and discussed. There were indications of a proline binding protein present, but no evidence of an isoleucine binding protein, in the osmotic shock supernatant fluid. The isoleucine binding-protein, or proteins, appeared as an integral part of the cytoplasmic membrane. The data were discussed in an attempt to clarify the mechanism of amino acid transport in P. aeruginosa and to define the role of the amino acid binding proteins in the phenomenon of active transport. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In ruminants the aerobic host animal is dependent on the anaerobic fermentation within the rumen to supply protein and energy. This anaerobic fermentation results in a rather constant ratio between protein and energy. In young growing animals and lactating animals the demand for protein is greater than the demand for energy. Plasma amino acid data indicate that protein could be limiting in these two situations. In the first phase of the study, plasma free amino acids, milk volume and milk components were determined at two week intervals throughout the lactations of two mature Ayrshire cows. The large amounts of protein that were secreted daily during the first third of lactation placed heavy demands on the animals for protein supplies. These demands were reflected in changes in plasma free amino acids during the lactation cycle. Lysine, leucine, isoleucine and methionine exhibited the greatest variation in response to the demands of lactation. A series of abomasal infusions of the above amino acids and others was undertaken to determine the importance of amino acids for the synthesis of milk protein. The infusions were grouped into three lots; those containing methionine, those containing isoleucine and those containing lysine. Effects observed during infusions containing methionine suggested that methionine itself had little effect on- protein production. Methionine did not alter the effects of other amino acids on milk protein synthesis. The responses noted when methionine was accompanied by branched chain amino acids or by lysine were typical of responses of branched chain amino acids or lysine alone. Infusions containing isoleucine and other branched chain amino acids caused an increase in milk protein concentration, a decrease in milk production and no increase in total protein production. Plasma amino acid data revealed that infusion of branched chain amino acids resulted in decreases in concentrations of the other plasma amino acids. The decrease in plasma amino acid concentration, the lack of response in milk protein production and the known effects of branched chain amino acids on insulin secretion suggest that insulin might be affecting the intake of amino acids by muscle tissue. When lysine-containing infusions were examined milk production was found to increase. Milk protein concentration remained constant resulting in an increase in total milk protein production. The most dramatic increase in milk and protein production occurred when lysine was infused alone. Infusions containing lysine did not result in decreases in plasma free amino acids. There was a general trend for all the essential amino acids to increase or remain at the same level during the infusion. Lysine appears to be the limiting amino acid for milk protein synthesis. It is possible that the increased supply of lysine from the abomasal infusion affected the amino acid metabolism in muscle in such a way as to free amino acids for milk protein synthesis and energy metabolism. / Land and Food Systems, Faculty of / Graduate

The development of a chemically defined artificial diet for Schistocerca gregaria (Forsk.) is described,. The diet that permitted the best growth of S. gregaria was used to determine the amino acid requirements of this animal. Further studies were performed to determine whether amino acids were required as nutrient per se or as phagostimulants. The moist artificial diets initially used in growth trials showed signs of deterioration after 20-days storage at -15°C. This deterioration was evidenced by reduced growth of animals on 20-day-old diets, and by the loss of ascorbic acid from such diets. Freeze-or oven-drying diets increased their storage life and their stability under experimental conditions. Growth trials showed that dried diets were stable for at least 2 months when stored in vacuum desiccators. Little ascorbic acid was degraded in diets kept under experimental conditions (30°C, 55% E.H.) for 2 days, suggesting that such diets could be kept under these conditions for at least 2 days without being replaced. S. gregaria showed poor growth on artificial diets containing either an ad hoc mixture of amino acids or a mixture of amino acids based on analysis of lettuce protein. However, growth of animals was improved by using an amino acid mixture based on analysis of cabbage proteins. Diets could be prepared mere rapidly if the vitamin solutions used in compounding the diets were replaced by vitamins triturated in sucrose. The physical properties of the diet were important, and fine-powder diets caused heavy mortality of S. gregaria hoppers.. Powder diets had to be formed into granules or tablets before they could be utilized by S. gregaria. Although few animals reached the adult stage on artificial diets, the best diet did allow S. gregaria hoppers to develop to the 5th instar (with a mean weight of approximately 550 mg) after 33-days growth. To determine which amino acids were essential for S. gregaria, the growth of animals reared on test-diets lacking an individual amino acid was compared with growth of animals on control diets containing the full complement of amino acids. If the removal of an amino acid had no effect on the growth of animals it was classed as an inessential amino acid; if it had an marked detrimental effect it was classed as an essential amino acid, and if it only had a marginally detrimental effect it was termed a semi-essential amino acid. The results of growth trials indicated that tyrosine, alanine, aspartate, glutamate, cystine, serine and proline were inessential amino acids for S. qreqaria, whereas lysine, phenylalanine, isoleucine, valine, threonine, tryptophan, leucine, histidine, methionine and arginine were essential, and glycine was semi-essential. Although the growth of animals on arginine deficient diets was poor enough to warrant arginine being classed as an essential amino acid, S. qreqaria's requirements for this amino acid did not appear to be as stringent as its requirements for the ether essential amino acids. Ihe semi-essential nature of glycine, and the suprisingly good growth of animals on arginine-deficient diets are discussed in relation to S. qreqaria1s amino-acid metabolism. It is suggested that the poor growth of animals on glycine-deficient diets is a result of glycine not being synthesized rapidly enough to meet S. gregaria’s metabolic requirements, and that the growth of animals on arginine-deficient diets is due to limited synthesis of this amino acid via the ornithine cycle. Feeding trials indicated that the failure of animals to show good growth on diets lacking any of the 10 essential amino acids was due to the reduced feeding activity of animals reared on these diets. Individual removal of any of the other 8 amino acids (i.e. the 7 inessential amino acids and glycine) had no effect on food consumption. The role of amino acids as phagostimulants is discussed in relationship to current theories concerning host-plant selection by phytophagous insects, and it is suggested that food selection is based on a learned-aversion response to the metabolic effects associated with the ingestion of an imbalanced ratio of nutrients. / Science, Faculty of / Zoology, Department of / Graduate

The growth of weanling rats receiving varying levels of methionine plus cystine in barley-based diets showed that 0.23-0.40% dry matter (DM) basis levels could not support optimal growth. No significant differences were detected in carcass composition or growth of rats receiving 0.45-0.70% DM methionine plus cystine dietary levels when either the cystine level was held constant at 0.20% DM in all diets or the methionine:cystine ratio was held constant at 2:1 and 1:1 in all diets. When either the dietary cystine concentration was held constant at 0.20% DM or the methionine:cystine ratio was held constant at 2:1 in all diets, the results indicated that cystathionine synthase activity was constant between 0.35 and 0.50% DM dietary methionine plus cystine. The activity of the enzyme was then inhibited, reaching minimum activity at the 0.60% DM dietary methionine plus cystine level. Thereafter, the activity increased to levels higher than the activity levels obtained between 0.35 and 0.50% DM dietary methionine plus cystine levels. These results indicate that 0.50% DM dietary methionine plus cystine is the limit for normal methionine metabolism. When either the dietary cystine concentration was held constant at 0.20% DM or the methionine:cystine ratio was held constant at 2:1 in all diets, the activity of N⁵-methyltetra-hydrofolate-homocysteine-methyltransferase (mTHF Enz.) was constant between 0.35 and 0.50% DM methionine plus cystine. The enzyme activity then increased, reaching maximum levels at the 0.60% DM methionine plus cystine level. Thereafter the enzyme activity was inhibited to levels corresponding to activities obtained between 0.35-0.50% DM dietary methionine plus cystine. The results indicate that normal methionine metabolism occurs up to the 0.50% DM methionine plus cystine in the diet. When the methionine:cystine ratio was held constant at 1:1 in all diets in which the methionine plus cystine concentration varied between 0.35 and 0.70% DM, cystathionine synthase activity did not respond to the varying levels of methionine plus cystine, whereas mTHF Enz. activity was inhibited progressively with increasing levels of dietary methionine plus cystine. The same results showed that increasing the dietary serine level from 0.38 to 0.58% DM depressed feed intake and the activities of both cystathionine synthase and mTHF Enz. in rats fed the 0.35-0.70% DM dietary methionine plus cystine range. The interaction between choline and methionine plus cystine when the methionine: cystine ratio was held at 1:1 in all diets showed that the 1200 mg choline chloride/kg DM diet inhibited mTHF Enz. activity and activated cystathionine synthase more than the 1000 mg choline chloride/kg DM diet. The pig trial, in which gilts were fed barley-based diets containing varying levels of methionine plus cystine, showed that the change in urinary urea-nitrogen excretion of pigs on test diets from the positive control diet, could be used as an indicator of methionine plus cystine requirements for optimal growth. Using this parameter, the requirement for methionine plus cystine for the 32.6±0.6 kg gilt was 0.55% DM on barley-based diets where cystine was held constant at 0.20% DM level. / Land and Food Systems, Faculty of / Graduate

The nature of this study involved the preparation of the coordination compounds of platinum (II) and four alpha-amino acids, namely arginine, histidine, lysine, and serine. A first analysis of the problem would seem to indicate that the preparations should be straightforward and not difficult. Bailar (3) stresses the great coordination tendency of the alpha-amino acids. Secondly, Volshtein (37) (38) (39) (40) (41) (41) and others have synthesized a number of platinum (II) complexes of the alpha-amino acids with apparently a minimum of difficulty in a majority of the preparations. FInally, a number of complexes of the amino acids under study have been prepared with other metals including cobalt, copper, chromium, and zinc.

In the world of living organisms there are thousands of separate and individual chemical reactions taking place in the course of their intermediary metabolism. These reactions are catalyzed by complex organic catalysts known as enzymes. One such reaction involves the deamination of amino acids in order that these amino acids may be used as fuel. This reaction takes place according to the equation [--see thesis for equation--] and is catalyzed by the enzyme amino acid oxidase. Most of the natural occuring amino acids belong to the 1 series. However, in the body there are found 1-amino acid oxidases and d-amino acid axidases both of which are stereo-specific and are without effect upon the amino acid of opposite stereochemical configurations of the two such groups of enzymes found in mammals the group which is by far the most active is the d-amino acid oxidase. These enzymes are capable of performing their catalytic activity outside of the cell which produce them. In studying the activity of various tissues for oxidase activity it is, therefore, common practice to separate the tissue from the animal, masserate the tissue to destroy all the cells and extract the enzymes from the masserated tissue. To such extract the individual amino acid may be added the oxidative deaminase observed in vitro. One of the difficulties in such experimentation consists in the measurement of the rate and extent of the reaction. This has previously been done by measuring the rate and extent of oxygen consumed or the rate and extent of the keto-acid production. Both of these methods present inherent weaknesses which make them unsuitable for accurate and extensive work. It was believed that a measure of the rate of ammonia production would be more accurate and a much easier determination to perform than either of the other two. It was toward this end that the research of this thesis was directed. By using the rabbit kidney tissue and various d-amino acids it was shown that deamination of these amino acids as measured by the rate fo ammonia production followed the same general pattern as reported by Krebs and his contemporaries as measured by oxygen consumption and keto-acid production. The ammonia thus produced was easily and accurately measured with Nessler's reagent by a photoelectric colorimeter. The tissue from several other rabbit organs were also examined for d-amino acid oxidase activity and considerable variation of such activity was shown in the study.

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