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.

Cysteine metabolism in cultured tobacco cells.

Transported l-[(35)S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The (35)S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[(35)S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the (35)S label present as compounds other than cysteine.The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH(4) (+). Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K(m) to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.

Cysteine transport into cultured tobacco cells.

Cysteine transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured on liquid B-5 medium was examined.Transport was linear with time or amount of tissue and had a pH optimum of 4.5. Cysteine transport over a wide concentration range was biphasic. The isotherm, for descriptive convenience, was divided into two segments both of which obeyed Michaelis-Menten kinetics. The Km for high affinity transport was in the range 1.7 x 10(-5)m(+/-0.17) while the Km for low affinity transport was in the range 3.5 x 10(-4)m(+/-0.13). Maximum velocities were 3 to 6 nmoles/g fresh weight/minute and 13 to 16 nmoles/g fresh weight/minute, respectively.Azide and 2,4-dinitrophenol caused more than 90% inhibition of net transport by either system. N,N'-Dicyclohexylcarbodiimide was not inhibitory while the inhibition by carbonylcyanide m-chlorophenylhydrazone was dependent on the cysteine concentration. Only those compounds that were inhibitory to transport caused significant efflux of labeled material from preloaded cells.Tobacco cells that had been preincubated in iodoacetamide or N-ethylmaleimide did not transport cysteine while similar treatments with dithiothreitol were only slightly inhibitory or had no effect on transport.Transport by either system was, to some extent, inhibited by all other tested amino acids and analogs. Alanine, methionine, and S-methyl cysteine were most effective in inhibiting cysteine transport. Both alanine and methionine were competitive inhibitors of cysteine transport by either system with inhibition constants that were similar to the Km for the particular system.

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis.

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38 degrees C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54 degrees C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36 degrees C. Temperatures below 36 degrees C or above 40 degrees C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34 degrees C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36 degrees C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38 degrees C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.

Interaction of heat and salt shock in cultured tobacco cells.

Cultured tobacco cells (Nicotiana tabacum L. var Wisconsin-38) developed tolerance to otherwise nonpermissive 54 degrees C treatment when heat-shocked at 38 degrees C (2 h) but not at 42 degrees C. Heat-shocked cells (38 degrees C) exhibited little normal growth when the 54 degrees C stress came immediately after heat shock and normal growth when 54 degrees C stress was administered 8 hours after heat shock. Heat shock extended the length of time that the cells tolerated 54 degrees C. Tobacco cells developed tolerance to otherwise lethal 2% NaCl treatment when salt-shocked (1.2% NaCl for 3 hours). The time course for salt tolerance development was similar to that of thermotolerance. Heat-shocked cells (38 degrees C) developed tolerance of nonpermissive salt stress 8 hours after heat shock. Alternatively, cells heat-shocked at 42 degrees C exhibited immediate tolerance to lethal salt stress followed by a decline over 8 hours. Radioactive methionine incorporation studies demonstrated synthesis of heat shock proteins at 38 degrees C. The apparent molecular weights range from 15 to 115 kilodaltons with a protein complex in the 15 to 20 kilodalton range. Synthesis of heat shock proteins appeared to persist at 42 degrees C but with large decreases in incorporation into selected heat shock protein. During salt shock, the synthesis of normal control proteins was reduced and a group of salt shock proteins appeared 3 to 6 h after shock. Similarities between the physiology and salt shock proteins/heat shock proteins suggest that both forms of stress may share common elements.

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.

Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin.

A cDNA clone (pTCB48) encoding a calmodulin-binding protein was isolated by screening a lambda ZAPII cDNA expression library constructed from cell cultures of heat-shocked tobacco (Nicotiana tabacum L. cv Wisconsin-38) with metabolically labeled [35S]calmodulin. Calmodulin gel overlay analysis indicated that pTCB48 generated major peptides of 53, 36, and 22 kD and two minor peptides of 37 and 16 kD that bound calmodulin in a Ca(2+)-dependent manner. Deletion analysis of pTCB48 indicated that these and the minor calmodulin-binding proteins resulted from the insert. A probe made from the cDNA insert recognized two bands with sizes of 2.1 and 1.8 kb on northern blot analysis. Both species of RNAs were undetectable in the control and were induced after 15 min of heat-shock treatment at 38 degrees C. The intensity of the two bands reached maximum after 1.5 h of heat-shock treatment. The cDNA clone was not full length; however, the complete sequence was determined by 5' rapid amplification of cDNA ends using nested antisense primers. The full-length cDNA contains 1648 bp and a single open reading frame of 1347 bp and is expected to encode a protein of approximately 50 kD. No significant homology with other reported genes and proteins was found. Structural predictions, deletion analysis, and gel overlay analysis suggested that the calmodulin-binding domain was a basic amphiphilic alpha-helix near the C terminus of the protein. The strong induction of the mRNA for this protein suggests a role for Ca2+/calmodulin-mediated process in the heat-shock response.

Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein.

A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys- Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM Mr in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (Ki = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide, which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. 1H NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.

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.

Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.

A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.