Purified gamma-glutamyl transpeptidases from tomato exhibit high affinity for glutathione and glutathione S-conjugates.

gamma-Glutamyl transpeptidases (gammaGTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (gamma-L-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two gammaGTases (I and II) with K(m) values for glutathione of 110 and 90 microM were purified 2,977-fold and 2,152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyze dipeptides and other tripeptides with N-terminal, gamma-linked Glu and the artificial substrates gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, including L-Met, L-Phe, L-Trp, L-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. gammaGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 M NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, Dye Matrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two gammaGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kD.

Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover.

Turnover rate is an important aspect of the regulation of plant processes by plant growth substances. To study turnover of indole-3-acetic acid (IAA), two [alpha]-methyltryptophan-resistant lines (MTR1 and MTR2) of Lemna gibba were generated by nitrosomethyl urea treatment of an inbred line derived from L. gibba G-3. In this report we describe: (a) the development of a selection system using this near isogenic line of L. gibba; (b) techniques for chemical mutation of the lines and selection for [alpha]-methyltryptophan resistance; and (c) the partial characterization of the selected lines. MTR lines contained 3-fold higher levels of anthranilate synthase activity. The enzyme in the MTR lines required higher levels of tryptophan for feedback inhibition. MTR lines also contained 8-fold higher levels of tryptophan, 3-fold higher levels of free IAA, and similar levels of total IAA compared to the inbred line. Turnover rates in the inbred and selected lines were calculated, using the first-order rate equation, based on the decrease over time in isotopic enrichment of I3C6-IAA introduced into L. gibba during a 1-h pulse period. Isotope enrichment in IAA was determined by using gas chromatography-mass spectrometry. Both MTR lines had an approximately 10-fold higher rate of IAA turnover than the parent inbred line.

Indole-3-Acetic Acid Biosynthesis in the Mutant Maize orange pericarp, a Tryptophan Auxotroph.

The maize mutant orange pericarp is a tryptophan auxotroph, which results from mutation of two unlinked loci of tryptophan synthase B. This mutant was used to test the hypothesis that tryptophan is the precursor to the plant hormone indole-3-acetic acid (IAA). Total IAA in aseptically grown mutant seedlings was 50 times greater than in normal seedlings. In mutant seedlings grown on media containing stable isotopelabeled precursors, IAA was more enriched than was tryptophan. No incorporation of label into IAA from tryptophan could be detected. These results establish that IAA can be produced de novo without tryptophan as an intermediate.

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid.

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [(15)N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[(15)N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[(15)N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.

Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture.

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with (13)C(6)-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR(1)) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR(1) has the same amount of DNA per nucleus as the parent line (PL). All jsR(1) cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR(1) at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR(1) had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR(1) and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR(1) had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity.

Comparison of a commercial ELISA assay for indole-3-acetic Acid at several stages of purification and analysis by gas chromatography-selected ion monitoring-mass spectrometry using a c(6)-labeled internal standard.

Quantitative analysis of indole-3-acetic acid (IAA) using selected ion monitoring gas chromatography-mass spectrometry (GC-MS) with (13)C(6)[benzene ring]-IAA as the internal standard was used to compare the quantitative accuracy of commercial enzyme-linked immunoabsorbent assay (ELISA) kits. Plant materials differed in the amount of purification required prior to use of ELISA for reliable estimates to be made. Purification similar to that obtained by at least one high performance liquid chromatographic (HPLC) step was generally necessary prior to ELISA analysis of plant materials. Additional levels of purification appeared to be required for some plant materials prior to HPLC in order to obtain an accurate estimate by ELISA techniques. In no case was it possible to obtain reasonable estimates of IAA from crude extracts or even from acidic fractions of extracts of plant tissues. GC-MS techniques provide a rapid and simple method for checking the validity of ELISA techniques. Quantitative GC-MS, or a similar technique that provides an independent quantitative validation, should, whenever possible, be applied to each new plant material under study if use of the ELISA is planned.

Gamma-actin: unusual mRNA 3'-untranslated sequence conservation and amino acid substitutions that may be cancer related.

beta-Actin mutations in chemically transformed human cell lines have been associated with tumorigenicity, an association consistent with other evidence suggesting that altered cytoskeletal proteins may have an important role in cancer initiation or progression. From a human promyelocytic leukemia cell line, we have isolated a gamma-actin cDNA clone with amino acid substitutions in a region highly conserved in the many actins analyzed. To our knowledge, this is the first example of a variant gamma-actin in a human neoplasm. A separate finding from the analysis of this clone is that the gamma-actin 3'-untranslated region is among the most highly conserved of all 3'-untranslated sequences so far reported, but is entirely different from the beta-actin 3'-untranslated region. The high degree of evolutionary conservation suggests that the 3'-untranslated regions of these two mRNAs have important and distinct functional roles that were already fully differentiated more than 100 million years ago. Mutations affecting four major cytoskeletal components have now been identified in human neoplastic cells. These findings suggest that mutated cytoskeletal genes may be members of a class of oncogenes, fundamentally different from both the nuclear-acting (e.g., myc and simian virus 40 large tumor antigen) and growth factor/receptor/protein kinase-related (e.g., sis, erbB, and ras) types of oncogenes.

mRNA species regulated during the differentiation of HL-60 cells to macrophages and neutrophils.

Using cDNA clone banks from differentiated and undifferentiated HL-60 promyelocytic leukemia cells, we have selected clones for genes which are regulated during this differentiation. Regulation of the corresponding mRNAs in HL-60 cells during both monocytic and neutrophilic differentiation was measured for 21 of these clones. The levels of mRNA hybridizing to some of these clones changed by more than 100-fold during differentiation. Unlike erythropoiesis or myogenesis, in which the synthesis of a few new proteins is synchronously regulated, mRNAs in differentiating HL-60 cells are asynchronously regulated, suggesting a complex series of regulatory events. About half of these regulation-selected clones contained repeat sequences, including both Alu and novel repeat families. Most of the regulated genes are members of extensive gene families.

C(6)-[benzene ring]-indole-3-acetic Acid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic Acid in plants.

Indole-3-acetic acid (IAA) labeled with (13)C in the six carbons of the benzene ring is described for use as an internal standard for quantitative mass spectral analysis of IAA by gas chromatography/selected ion monitoring. [(13)C(6)]IAA was compared to the available deuterium labeled compounds and shown to offer the advantages of nonexchangeability of the isotope label, high isotopic enrichment, and chromatographic properties identical to that of the unlabeled compound. The utility of [(13)C(6)]IAA for measurement of endogenous IAA levels was demonstrated by analysis of IAA in Lemna gibba G-3.

Synthesis and turnover of the light-harvesting chlorophylla/b-protein inLemna gibba grown with intermittent red light: possible translational control.

Lemna gibba L. G-3 plants grown heterotrophically in the dark with intermittent red light (2 min every 8 h) contain a substantial amount of translatable mRNA encoding the light-harvesting chlorophyll (Chl)a/b-protein. However, very little [(35)S]methionine is incorporated into the apoproteins during a 1-h labeling period in the dark in these plants compared to plants grown in continuous white light. The Chla/b-protein mRNA is found to be associated with functioning polysomes in plants grown in the dark with intermittent red illumination (R plants). The small amounts of the apoproteins which are synthesized by these plants are found in the membrane fraction; neither the mature apoproteins nor their precursor(s) can be detected immunologically in the soluble fraction. The protein does not accumulate in these plants. Pulse-chase experiments with the R plants demonstrate that the newly synthesized apoproteins have a half-life of about 10 h in the dark. This turnover is not sufficient to explain the observed 20-fold difference in [(35)S]methionine incorporation into the apoprotein between white-light-grown and R plants. We therefore suggest that the synthesis of the Chla/b-apoproteins can be regulated by a light-dependent step at the level of translation, and that this regulation occurs after the initiation of translation.