A mixed layer for separation of amino acids by thin-layer chromatography

Turner, N. A.; Redgwell, Robert J.

doi:10.1016/s0021-9673(01)91273-7

Cited by 49 publications

(8 citation statements)

References 7 publications

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“…Amino acids were identified and quantified against 22 amino acid standards using thin-layer chromatography with single-and double-dimension solvent systems [24][25][26] and ninhydrin staining. Glutamate and glutamine were identified as the two major amino acids that were present.…”

Section: Assaysmentioning

confidence: 99%

Composition of Luminal Fluid Secreted by the Seminiferous Tubules and After Reabsorption by the Extratesticular Ducts of the Japanese Quail, Coturnix coturnix japonica1

Clulow¹,

Jones²

2004

Biology of Reproduction

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The present report examines the composition of luminal fluid in the seminiferous tubule (STF), rete testis (RTF), and ductus epididymidis of the Japanese quail (Coturnix coturnix japonica). This subject is of particular interest, both because the reproductive ducts are intra-abdominal and because sperm production is more rapid in birds than in mammals. It was interpreted that micropuncture samples of STF contain varying amounts of contamination with intracellular solute, particularly K and protein. The concentration of solute in samples was correlated with packed cell volume (spermatocrit), and when the latter was used to assess estimates of solute concentration in STF, the magnitude of the estimates were much the same as determinations in RTF. Consequently, it is concluded that the fluid entering the rete testis of the quail is the primary secretion of the seminiferous tubules. The composition of RTF in the quail was determined to be 148 mM Na, 126 mM Cl, 9.8 mM K, 2.7 mM Mg, 1.4 mM Ca, 2.1 mM glutamate, 3.4 mM glutamine, 20.2 mM bicarbonate, 1.8 microg microl(-1) of protein, pH 7.34, and 310 mmol kg(-1), and it is significantly different from the composition of blood plasma. Estimates of solute output by the testis and reabsorption by the extratesticular ducts indicate, first, that most of the solutes secreted into the seminiferous tubules are subsequently reabsorbed from the extratesticular ducts and, second, that sufficient solute of testicular origin (except for protein) exists to account for the concentrations of solutes throughout the lumen of the duct system. Changes in the concentration of solute in the extratesticular ducts probably result from different reabsorption rates of solute and water. The composition of fluid from the distal end of the ductus epididymidis was 133 mM Na, 125 mM Cl, 25 mM K, 1.0 mM Mg, 0.3 mM Ca, 6.7 mM glutamate, 4.0 mM glutamine, 19.5 mM bicarbonate, 6.0 microg microl(-1) of protein, pH 7.33, and 335 mmol kg(-1), and it is significantly different from those of RTF and blood.

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Section: Assaysmentioning

confidence: 99%

Composition of Luminal Fluid Secreted by the Seminiferous Tubules and After Reabsorption by the Extratesticular Ducts of the Japanese Quail, Coturnix coturnix japonica1

Clulow¹,

Jones²

2004

Biology of Reproduction

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“…Chromatography and Autoradiography of 14C-Amino Acids Amino acid fractions from leaf blades of the infected and uninfected plants were separated by two-dimensional chromatography on mixed cellulose/silica gel thin-layer plates prepared according to the procedure of Turner and Redgwell (23). The solvent system in the first direction was phenol/ water (80:20, w/v) and in the second direction it was nbutanol/acetic acid/water (5:1:4, v/v, upper phase).…”

Section: Co2 Assimilationmentioning

confidence: 99%

“…Outwardly, such infections cannot be detected despite extensive intercellular growth within the plant; the fungus is not a pathogen. Initially recognized as an essential causal factor in the toxicity of tall fescue to cattle (3,4,9), infected stands of tall fescue may be enhanced in their ability to survive under stressful environments (2,5,23). Thus, the endophytic fungus has considerable agronomic and ecological significance on tall fescue.…”

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confidence: 99%

Effects of the Fungal Endophyte Acremonium coenophialum on Nitrogen Accumulation and Metabolism in Tall Fescue

Lyons¹,

Evans²,

Bacon³

1990

Plant Physiol.

133

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Infection by the fungal endophyte Acremonium coenophialum affected the accumulation of inorganic and organic N in leaf blades and leaf sheaths of KY 31 tall fescue (Festuca arundinacea Schreb.) grown under greenhouse conditions. Total soluble amino acid concentrations were increased in either the blade or sheath of the leaf from infected plants. A number of amino acids were significantly increased in the sheath, but only asparagine increased in the blade. Infection resulted in higher sheath NH4+ concentrations, whereas N03-concentrations decreased in both leaf parts. The effects on amino acid, N03-, and NH4+ concentrations were dependent upon the level of N fertilization and were usually apparent only at the high rate (10 millimolar) of applica- feeding (2,4,5,11,21). Many effects similar to those in endophyte-infected tall fescue have also been demonstrated in other endophyte-infected species of fescue and in perennial ryegrass (Lolium perenne L.) ( 14, 21).The underlying physiological basis for the various effects on tall fescue which result from this endophyte infection, and the extent to which the grass and fungus are responsible, are largely unknown. Two lines of evidence suggest that at least N metabolism is one aspect that may be affected. The first is from greenhouse studies which show that high rates of N fertilization increased the concentrations of one class of endophyte-synthesized N bases, ergot alkaloids, in endophyteinfected tall fescue (16). Second, other classes of nitrogenous metabolites that are insect feeding deterrents are associated with endophyte infection. Furthermore, the expression of cattle toxicoses has been quantitatively related to rates of N fertilization (3,7,21). Thus, in tall fescue, both fungal and host N metabolism may be contributing factors to various characteristics which are peculiar to the infected plant.Because of the possible involvement of N metabolism in these important aspects of the tall fescue-endophyte association, comparative studies were made of infected and uninfected plants under greenhouse conditions to determine whether N metabolism is affected by the endophyte. Aspects considered in this study included the accumulation of inorganic and organic nitrogenous compounds, the activities of major enzymes of N assimilation, and the metabolism of amino acids. A

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“…This evidence suggests that not only was saccharopine formed in the assay, but the measure of NADPH oxidation dependent on lysine and a-ketoglutarate is a reasonable estimate of the lysine-ketoglutarate reductase activity. Further evidence for the identity of saccharpine as a product of the reaction was obtained by TLC using silica-cellulose mixed bed plates (15) with authentic saccharopine as marker. In all three solvent systems tested (Table II), the ninhydrin-positive reaction product ran with saccharopine.…”

Section: Resultsmentioning

confidence: 99%

Lysine-Ketoglutarate Reductase Activity in Developing Maize Endosperm

Arruda¹,

Sodek²,

Silva³

1982

Plant Physiol.

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Lysine-ketoglutarate reductase activity was detected and characterzed in the developing endosperm of maize (Zea mays L). The enzyme showed specificity for its substrates: lysine, a-ketoglutarate, and NADPH. Formation of the reaction product saccharopine was demonstrated. The pH optimum of the enzyme was cdose to 7, and the Km for lysine and aketoglutarate were 5.2 and 1.8 millmolar, respectively. between 25 and 70%o saturation was collected and taken up in 2.5 ml of extraction buffer. After desalting on a Sephadex G-25 column (1.5 x 16 cm), the protein fraction was assayed for lysineketoglutarate reductase activity using conditions based on those described by Hutzler and Dancis (7). The assay mixture contained K-phosphate (pH 7.0; 300 ,umol), L-lysine (50 ,pmol), a-ketoglutarate (25 ,umol), NADPH (300 nmol), and enzyme (0.1-0.2 mg protein) in a total volume of 3 ml. The mixture was incubated at 30°C, and oxidation of NADPH was monitored in a spectrophotometer at 340 nm.Lysine-ketoglutarate reductase catalyzes the following reaction: lysine + a-ketoglutarate + NADPH -* saccharopine + NADP+ The enzyme has been characterized in human and animal tissues (3,7,8) where it is believed to catalyze the first step of lysine catabolism (4). A similar reaction involving NAD is catalyzed by saccharopine dehydrogenase, an enzyme found in yeast and fungi where the reverse of the above reaction is considered to be the final step of lysine biosynthesis (5,6,12,16). Thus, in some organisms saccharopine is an intermediate of lysine biosynthesis while in the others it appears to be involved in lysine breakdown. Actually, several pathways for lysine catabolism are known (11), but in higher plants no general pathway has been established. Nevertheless, tracer studies with barley seedlings have provided strong evidence for the pathway involving saccharopine as intermediate (10), suggesting that this route may be important for lysine breakdown in cereals at least. However, we are unaware of any studies with enzymes in higher plants in support of this pathway.In this report, we describe the characterization of lysine-ketoglutarate reductase in developing maize endosperm, a tissue known to degrade lysine extensively (14). MATERIALS AND METHODS RESULTS AND DISCUSSIONThe data in Table I show that partially purified extracts of maize endosperm oxidized NADPH in the presence of lysine and a-ketoglutarate. In the absence of one or both of these substrates, the oxidation of NADPH was minimal. Specificity for the substrates is indicated by the fact that several amino acids, including closely related structures such as L-ornithine and D-lysine, would not substitute for L-lysine, nor would oxaloacetate or pyruvate substitute for a-ketoglutarate. NADH would not replace NADPH as electron donor, as was found for the enzyme from human liver (7).To confirm that the activity being measured was truly lysineketoglutarate reductase, it would be essential to demonstrate the formation of saccharopine in the assay. This was attempted using I4CJlysine...

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A mixed layer for separation of amino acids by thin-layer chromatography

Cited by 49 publications

References 7 publications

Composition of Luminal Fluid Secreted by the Seminiferous Tubules and After Reabsorption by the Extratesticular Ducts of the Japanese Quail, Coturnix coturnix japonica1

Composition of Luminal Fluid Secreted by the Seminiferous Tubules and After Reabsorption by the Extratesticular Ducts of the Japanese Quail, Coturnix coturnix japonica1

Effects of the Fungal Endophyte Acremonium coenophialum on Nitrogen Accumulation and Metabolism in Tall Fescue

Lysine-Ketoglutarate Reductase Activity in Developing Maize Endosperm

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