Two isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, denoted DS-Mn and DS-Co, were identified following DEAE-cellulose chromatography of crude extracts prepared from suspension-cultured cells of Nicotiana silvestris. The strikingly different properties of the isozymes allowed the development of assays for the selective detection of either isozyme in samples containing a mixture of the two. The DS-Mn isozyme required the sulfhydryl reductant, dithiothreitol, for activity and was stimulated by manganese. Activation by dithiothreitol was slow relative to catalysis, accounting for a hysteretic progress curve that was observed when reactions were started with inactive enzyme. The DS-Co isozyme was inhibited by dithiothreitol and required a divalent cation for activity. At optimal cation concentrations of 10 millimolar (magnesium), 0.5 millimolar (cobalt), and 0.5 millimolar (manganese), relative activities obtained were 100, 85, and 20, respectively. The substrate saturation curves with respect to erythrose 4-phosphate differed markedly when the two isozymes were compared. As little as 0.5 millimolar erythrose 4-phosphate saturated DS-Mn, whereas a 10-fold higher concentration was needed for saturation of DS-Co. The pH optimum of DS-Mn was 8.0, while that of the DS-Co isozyme was 8.6. Leaves of both N. silvestris and spinach also exhibited the DS-Mn/ DS-Co isozyme arrangement, and the subcellular location of DS-Mn was shown to be the chloroplast compartment. By application of the differential assays for DAHP synthase isozymes, various monocotyledonous and dicotyledonous plants yielded data indicating the general presence of the DS-Mn/DS-Co isozyme pair in higher plants.biosynthesis and the separate subcellular location of such isozymes is just developing (16,17). The best studied example is chorismate mutase, shown in Nicotiana silvestris to exist as two isozymes (11,12): an allosterically controlled species (CM-1) being located in plastids and an unregulated species (CM-2) being located in the cytosol (7). Two isozymes of DAHP synthase were identified in Vigna radiata (23,24), and were denoted DAHP synthase-Mn (stimulated by, but not requiring Mn) and DAHP synthase-Co (requiring Co, Mg, or Mn for activity). This pair of DAHP synthase isozymes was also isolated from leaves of N. silvestris (9). When assay conditions were optimized for either isozyme, the activity of the remaining isozyme was barely detectable.The markedly distinct properties of DS-Mn and DS-Co from N. silvestris have been exploited to define assay conditions for exclusive assay of either one of the two isozymes in mixtures. A reliable methodology to discriminate DAHP synthase isozymes in crude extracts is particularly valuable for subcellular localization studies as well as assessment of the variation in isozyme levels in response to developmental and regulatory changes. Although DAHP synthase from a variety of plants has been studied (14,15,(20)(21)(22)26), particular assay conditions employed would have favored in mo...

The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by L-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by L-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.

Two isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase are partitioned into plastid (DS-Mn) and cytosolic (DS-Co) compartments of at least several higher plants (RA Jensen 1986 Rec Adv Phytochem 20: 257-258 cytosolic isozyme (DS-Co) in Nicotiana silvestris (3, 7). A rationale and supporting evidence that part or all of the aromatic amino acid pathway is present in both subcellular locations of higher plants have been presented (9, 10).A common subculture routine for fast-growing plant cells in suspension culture is a regimen in which stationary-phase cells are diluted fivefold into fresh medium to initiate a new growth cycle. A lag phase of 1 to 2 d is followed by about 3 d of exponential growth with a doubling time of about 42 h for N. silvestris at room temperature. In contrast to growth cycles routinely obtained with microorganisms where many generations of balanced exponential growth follow high dilution subculturing, less than two generations of truly exponential growth separate lag and stationary phases in the low dilution routine for plant cell subculture. This procedural difference is primarily because microorganisms do not exhibit the minimal-density requirement for growth that is characteristic of cultured plant cells. Thus, in plant subcultures the carryover of enzymes synthesized during stationary and/or lag phase may produce flawed characterizations of the apparent nature of exponential-phase cells.Separate populations of cells, derived from line ANS-1 of N. silvestris (2), and denoted EE cells have been maintained in a routine of uninterrupted exponential growth for up to 2 years. Such EE cells are expected to be free of significant carryover of enzymes and metabolites that may be preferentially formed in stationary-phase physiology, thereby approaching steady-state levels of macromolecules that define balanced growth. In the present study we have determined the levels of plastid and cytosolic isozymes of 3-deoxy-D>arabino-heptulosonate 7-P synthase during the progression of cells through lag, exponential, and stationary phases of a growth cycle, and in turn compared these levels to the quasisteady-state levels present in cells maintained continuously in exponential phase. MATERIALS AND METHODSGrowth Conditions and Cell Populations. Suspension-cultured cells were grown as previously described (2)

A plastid‐localized isozytne of 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase, denoted DS‐Mn, has been identified in a number of higher‐plant species. Parallel characterizations were made of DS‐Mn from Spinacia oleracea leaf tissue, Solanum tuberosum tubers, and Nicotiana silvestris suspension culture as sources of enzyme from plant materials which vary in phytogeny, developmental and tissue state, and physiological state. A highly conserved property of DS‐Mn is a transition between inactive and active states, mediated by DTT as a hysteretic activator. A procedure for isolation of DS‐Mn in the labile, inactive state is given. The process of activation appears to exhibit a higher pH optimum than the catalytic optimum. DTT‐containing preparations are very stable. The enzyme characteristically exhibits stimulation by Mn++ in the range of 45–50%, relatively high affinity for erythrose‐4‐phosphate (E4P), dramatic substrate inhibition above about 0.5mol m−3 E4P, sigmoid substrate saturation curves for both E4P and phosphoenolpyruvate, and inhibition by L‐arogenate (competitive against E4P and non‐competitive against PEP). DS‐Mn has a relatively high temperature optimum in the range of 45–50°C. Enzyme activity was lost when bound metal was stripped away by EDTA treatment. Reconstitution of the native‐enzyme level of activity was obtained with Ca++, and additional stimulation was achieved with Mn++. DS‐Mn control by L‐arogenate in the chloroplast is proposed as one key circuit in an overall pattern of allosteric control for the entire pathway of aromatic amino acid biosynthesis. This pattern is called sequential feedback inhibition. The potential for modulation of this control system by environmental cues induced by light‐dark transitions is discussed.

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