In-vivo evidence for metabolic control of amino-acid and protein synthesis by exogenous lysine and threonine in Mimulus cardinalis.

Young developing seedlings of Mimulus cardinalis Douglas (Scrophulariaceae) were grown in Hoagland's medium supplemented with lysine (LYS; 1.0 mM), threonine (THR; 1.0 mM), LYS+THR (1.0 mM ea.) or LYS+THR+methionine (MET; 1.0-1.0-0.1 mM, respectively). LYS or THR reduced the total amount of protein in the developing seedlings and when added in combination completely inhibited the accumulation of protein after 7 days. The combination of LYS+THR+MET enhanced the accumulation of protein. When MET was added to seedlings grown on LYS+THR for 9 days, the rate of protein accumulation reverted to normal. Studies using [(14)C]leucine demonstrated similar inhibitory patterns on protein synthesis. The effects of LYS and THR on the distribution of [(14)C]aspartate into the aspartate amino acids including MET were studied. LYS and THR, singly and synergistically when in combination, inhibited the synthesis of MET. Thus the simultaneous application of LYS and THR can be interpreted as producing an artificially induced MET auxotroph. These results indicate that LYS and THR act singly and together in vivo to inhibit the accumulation of soluble LYS, MET and THR in a manner indicative of end-product inhibition. Indirect evidence for end-product control of alanine, arginine, glycine, phenylalanine, proline, tryptophan, tyrosine and valine is also given.

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT.

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.

Partial purification and characterization of dihydrodipicolinic Acid reductase from maize.

Dihydrodipicolinic acid reductase, an enzyme which catalyzes the pyridine nucleotide-linked reduction of dihydrodipicolinic acid to tetrahydrodipicolinic acid in the biosynthetic pathway leading to l-lysine, has been partially purified from maize (Zea mays cv Pioneer 3145) kernels. The crude maize extract and the partially purified enzyme were assayed for dihydrodipicolinic acid reductase by their ability to restore the capability of crude extracts of a mutant Escherichia coli (CGSC 4549; defective in dihydrodipicolinic acid reductase) to synthesize diaminopimelic acid from aspartic acid and pyruvic acid.In a study of its properties, the Michaelis constant obtained for dihydrodipicolinic acid was 4.3 x 10(-4) and for NADPH the K(m) was 4.6 x 10(-5). The enzyme had a pH optimum close to 7 and was much more temperature labile than the bacterial enzyme. Its molecular weight was 8.0 x 10(4).Several compounds, viz., alpha-picolinic acid, l-pipecolic acid, isophthalic acid, and isocinchomeronic acid, with structures similar to dihydrodipicolinic acid inhibited the reductase reaction. Dipicolinic acid, the most potent of these inhibitors, acted as a competitive inhibitor with a K(i) of 9.0 x 10(-4). The competitive inhibition of the reductase reaction by dipicolinic acid (oxidized dihydrodipicolinic acid) suggests that the substrate for this enzyme was in the ring rather than the open chain form. Oxidized pyridine nucleotide inhibited the activity slightly.

Effect of activated charcoal on callus growth and shoot organogenesis in tobacco.

Incorporating activated charcoal (AC) in culture media has been shown to affect growth and development of various organisms. Since AC stimulates the development of tobacco haploid plantlets from cultured anthers, research was conducted to determine the effect of activated charcoal on pith-derived callus growth and shoot development in Nicotiana tabacum cv. Wisconsin 38. Our results indicate that the hormones required for callues growth and shoot development in Wisconsin-38 tobacco are adsorbed by AC, thereby inhibiting callus growth and prohibiting shoot development. This effect was observed even when AC was removed from the medium by filtration prior to culturing the callus.

Amino Acid Transport into Cultured Tobacco Cells: II. EFFECT OF CALCIUM.

The effects of calcium ions on lysine transport into cultured Wisconsin-38 tobacco cells were examined. In the presence of calcium, lysine was transported at a relatively low rate for 30 to 40 minutes followed by a period of increasing rates and subsequent stabilization at a higher rate after 2 to 3 hours. In the absence of calcium, transport was uniformly low.Time-dependent stimulation of lysine transport rate was observed after the cells had been preincubated in calcium-containing media. Similar treatments also resulted in the stimulated uptake of a variety of other amino acids, organic compounds, and sulfate. The stimulation of lysine uptake was apparently not due to nutrient starvation.Lysine transport was not stimulated in a time-dependent fashion by K(+), La(3+), Mg(2+), or Mn(2+). Cells with stimulated transport rates continued to exhibit high rates when washed with calcium-containing media followed by transport in calcium-containing media. All other cation wash treatments were inhibitory, although magnesium treatments resulted in partial preservation of stimulated transport rates. Cycloheximide inhibited the calcium/time-dependent stimulation of lysine transport and caused the stimulated rate to decay.The initial experimental treatments or the culture conditions may represent some form of shock that alters the membrane transport mechanism, thus reducing transport. The observed calcium/time-dependent stimulation may require protein synthesis and represents damage repair.

Lysine-methionine-threonine interactions in growth and development of Mimulus cardinalis seedlings.

Plants of Mimulus cardinalis Douglas (Scrophulariaceae) were grown in axenic culture from seed for 28 days on a minimal salts medium supple-mented with L-lysine, L-methionine, L-threonine, L-isoleucine, DL-or L-homoserine or DL-homocysteine, alone or in combinations ranging from 5 to 1000 µM. Abnormal growth was observed at the higher concentrations of all these aminoacidsexcept homocysteine. The lysine inhibition was significantly reduced by methionine, homocysteine or isoleucine. The threonine inhibition was significantly reduced by methionine or homocysteine. A combination of lysine and threonine at 1 MM was lethal. This synergistic effect was prevented when methionine, homoserine or homocysteine were added to the lysine-threonine combination. These results can be explained in terms of end-product control of aspartokinase and homoserine dehydrogenase by lysine and threonine, respectively, in the biosynthetic pathway to these aspartic-acid-derived amino acids.

Amino-acid transport into cultured tobacco cells : III. Arginine transport.

Arginine transport in suspension-cultured cells of Nicotiana tabacum L. cv. Wisconsin-38 was investigated. Cells that were preincubated in the presence of Ca(2+) for 6 h prior to transport exhibited stimulated transport rates. After the preincubation treatment, initial rates of uptake were constant for at least 45 min. Arginine accumulated in the cells against a concentration gradient; this accumulation was not the result of exchange diffusion. Arginine uptake over a concentration range of 2.5 µM to 1 mM was characterized by simple Michaelis-Menten kinetics with a Km of 0.1 mM and a Vmax of 9,000 nmol g(-1) fresh weight h(-1). Transport was inhibited by several compounds including carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide. Inhibition by these compounds was not the result of increased efflux resulting from membrane damage. A variety of amino acids and analogs, with the exception of D-arginine, inhibited transport, indicating that arginine transport was mediated by a general L-aminoacid permease. Competition experiments indicated that arginine and lysine exhibited cross-competition for transport, with Ki values similar to respective Km values. Arginine transport and low-affinity lysine transport are probably mediated by the same system in these cells.