Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P⁺) and nonpromotable (P⁻) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P⁺ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO₄ precipitated and used to transfect the P⁻ cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10⁻⁸ M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P⁻ JB6 Cl 30 cells yielded 40 to 50 colonies per 10⁵ cells. In contrast, transfection of Cl 30 cells with “P⁺ DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10⁵ cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P⁺ DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P⁺ lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P⁺ DNA, no evidence for acquisition of promotability was obtained. P⁻ JB6 Cl 25, like Cl 30, also permitted expression of transfected P⁺ DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P⁺ DNA and requires recipient cells of the appropriate phenotype for expression.     

History of immunology. Development of the concept of immunologic specificity, I

The induction of differentiation in human malignant T-lymphoblastic cell lines MOLT-3 and Jurkat by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined using the monoclonal antibodies OKT3, OKT4, OKT6, and OKT8 which are known to react with human T-cell differentiation antigens. It was found that in the presence of nanomolar concentrations of TPA the proportion of OKT3⁺ (mature T-cell marker) cells increased while the proportion of OKT4⁺, OKT6⁺, and OKT8⁺ (relatively immature T-cell markers) cells decreased. These changes in the distribution of the OKT antigens in MOLT-3 cells were found to be more prominent with MOLT-3 cells than when the Jurkat cells were used. In studies using a double labeling approach it was found that although the OKT3⁺ and E-rosette-positive (E⁺) cells appeared to belong to the same subpopulations of MOLT-3 cells, the OKT3 antigen was probably not related to the receptor for sheep erythrocytes because adsorption of the OKT3 antibody did not block E-rosette formation. Studies using the DNA synthesis inhibitor, arabinosylcytidine (ara-C) also indicate that DNA synthesis was not required for the induction of more mature T-cell antigens in the malignant T-cell lines by TPA. These studies, taken together with our earlier reports, support the conclusion that namomolar concentrations of TPA can induce differentiation in these malignant T-cell lines. Furthermore we have shown that the T-cell hybridoma antibodies are useful markers to detect differentiation changes in human T cells.     

Pristane induced gene activation.

Studies were performed to examine the effects of 2,6,10,14-tetramethyl pentadecane (pristane) versus 12-O-tetradecanoylphorbol 13-acetate (TPA) on the activation of the CAT gene under the regulatory control of viral promoter/enhancer elements transfected into NIH-3T3, CV-1 and COS-7 cells. The results of these studies demonstrated that (1) pristane or TPA induced trans-activation of SV2cat, HIVcat, RSVcat and MMTVcat in cells transfected with each respective plasmid construct, (2) only pristane induced activation of pA10cat and pOSP/11 and (3) neither TPA nor pristane trans-activated pSV0cat. Furthermore, treatment with either pristane or TPA elicited changes in the morphology of each of the cell lines. Collectively these results indicate that pristane is a potent inducer of gene expression and exhibits similar characteristics as the tumor promoter, TPA.     

Nicardipine-sensitive enhancement of high K+-evoked dopamine release in PC12 cells pretreated with 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on stimulus-evoked dopamine release were studied in PC12 cells. Pretreatment of the cells with TPA resulted in an enhancement of dopamine release which could be further stimulated by high concentrations of K⁺, A23187, but not with carbamylcholine. TPA-dependent, high-K⁺-evoked enhancement of dopamine release was studied in detail: a maximum release was observed (169% of control) in response to 50 mM KCl upon treatment with 10⁻⁷ M TPA for 5 min at 37°C. This enhancement of dopamine release was associated with the concomitant reduction of the concentration rise of intracellular Ca²⁺ ([Ca²⁺]_i) induced by a high concentration of K⁺ monitored by a fluorescent indicator, fura2. Thus, these data provide an example for alteration in the efficiency of stimulus-secretion coupling as pointed out in our previous paper. Moreover, we have shown that nicardipine, CdCl₂, and CoCl₂ inhibit high-K⁺-evoked dopamine release more effectively in TPA treated cells than that of untreated cells, and that the TPA-dependent, high-K⁺-evoked dopamine release observed in TPA treated cells is completely abolished by the presence of nicardipine, Cd²⁺ or Co²⁺, but is only partially inhibited in the presence of verapamil. These relevant findings suggest the possible involvement of protein kinase C in regulating the efficiency of a high-K⁺-evoked dopamine release through the modification of nicardipine-sensitive Ca²⁺ channels.     

Responses of Preneoplastic Epidermal Cells to Tumor Promoters and Growth Factors: Use of Promoter-Resistant Variants for Mechanism Studies

The JB6 mouse epidermal cell model system is being used to study the mechanism of promotion of transformation. Promotion of anchorage independence in JB6 cells occurs in response to second-stage but not first-stage promoters, and is inhibited by inhibitors of second-stage not first-stage promotion. A number of variants that are resistant to the phorbol diester TPA have been derived. Some are resistant to plateau density mitogenic stimulation by TPA; others are resistant to promotion of anchorage independence by TPA. Some of the mitogen-resistant variants were promotable by TPA, thus ruling out a requirement for TPA mitogenesis in promotion of transformation in JB6 cells. TPA promotable clones were also sensitive to mezerein and EGF while the TPA nonpromotable variants were also resistant to mezerein and EGF, suggesting that sensitivity to promoters in these JB6 cells is determined at a level distal to receptor binding. Promotion sensitivity did not require available EGF receptors since two TPA promotable variants were EGF receptorless. The mitogenic response of JB6 cells to TPA may however be mediated by EGF since four of four mitogen-resistant variants showed low to zero levels of EGF binding. Tumor promoting phorbol esters produce specific changes in cellular gangliosides. Certain of these changes occur in promotable but not nonpromotable variants of JB6 cells, suggesting that ganglioside changes may be involved in the process of promotion of transformation.     

Plasticity of Migrating CD1b+ and CD1b- Lymph Dendritic Cells in the Promotion of Th1, Th2 and Th17 in Response to Salmonella and Helminth Secretions

Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b⁺ L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b⁺ L-DCs. Salmonella infection reduced the constitutive expression of TNF-α and IL-4 mRNA in CD1b^- L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b⁺ or CD1b^- L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-γ and IL-10 genes, in response to Salmonella-driven CD1b⁺ L-DCs, whereas a preferential IL-10, IFN-γ and IL-17 gene expression was observed in response to Salmonella-driven CD1b^- L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4⁺ and CD8⁺ T cells was observed after stimulation of CD1b⁺ and CD1b^- L-DCs with helminth secretions. These results show that mature conventional CD1b⁺ L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b^- L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.     

12-O-tetradecanoylphorbol-13-acetate and UV radiation-induced nucleoside diphosphate protein kinase B mediates neoplastic transformation of epidermal cells

The molecular changes associated with early skin carcinogenesis are largely unknown. We have previously identified 11 genes whose expression was up- or down-regulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin keratinocyte progenitor cells (Wei, S.-J., Trempus, C. S., Cannon, R. E., Bortner, C. D., and Tennant, R. W. (2003) J. Biol. Chem. 278, 1758-1768). Here, we show an induction of a nucleoside diphosphate protein kinase B (NDPK-B) gene in response to TPA or UV radiation (UVR). TPA or UVR significantly induced the expression of NDPK-B both in vivo hyperplastic mouse skin and in vitro mouse JB6 Cl 41-5a epidermal cells. Indeed, this gene was also up-regulated in TPA or UVR-mediated skin tumors including papillomas, spindle cell tumors, and squamous cell carcinomas, relative to adjacent normal skins. Functional studies by constitutive expression of nm23-M2/NDPK-B in TPA susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b preneoplastic epidermal cell lines showed a remarkable gene dosage-dependent increase in foci-forming activity, as well as an enhancement in the efficiency of neoplastic transformation of these cells in soft agar but no effect on proliferation in monolayer cultures. Interestingly, stable transfection of the nm23-M2/NDPK-B del-RGD or G106A mutant gene in JB6 Cl 41-5a cells selectively abrogated NDPK-B-induced cellular transformation, implicating a possible Arg105-Gly106-Asp107 regulatory role in early skin carcinogenesis.     

Induction of human malignant T-lymphoblastic cell lines MOLT-3 and jurkat by 12-O-tetradecanoylphorbol-13-acetate: Biochemical,physical, and morphological characterization

The process of induction of human malignant T-lymphoblastic cell line MOLT-3 by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined. It was found that the induction process by TPA, which included increase in cells with receptors to sheep red blood cells (E-rosette positive-E⁺) and decrease in the levels of the marker enzyme terminal deoxynucleotidyl transferase (TdT) was not affected by the presence of DNA synthesis inhibitor arabinofuranosylcytosine (Ara-C). The exposure time to TPA required to elicit these changes was found to be short, in the order of 1 hour or less. The kinetics of the increase in E⁺ cells, decrease in the levels of TdT in these cells, or decrease in the ability to proliferate as measured by colony formation were similar with exposure to TPA for 1, 6, 24, or 96 hours. We have examined the effect of antitumor promoter compounds on their ability to block induction of MOLT-3 cells by TPA. Results indicated that none of these compounds, dexamethasone, antipain, retinoic acid, and L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), was effective in reducing the number of E⁺ cells induced by TPA. Examination of three other leukemic T-cell lines indicated that, in addition to MOLT-3, the leukemic T-cell line Jurkat also responded to TPA, whereas two other leukemic T-cells lines CCRF-CEM and CCRF-HSB-2 did not. Certain physical and morphological changes were also observed after stimulation of MOLT-3 cells and Jurkat cells by TPA. We found that, following the addition of TPA, the cell volumes of MOLT-3 cells decreased from an average of 1150 μm³ to about 500 μm³, whereas those of Jurkat were reduced to about 700 μm³ from 1100 μm³. Electron microscopic studies of these lymphoblasts also revealed that after treatment with TPA the induced cells were generally smaller in size with increase in the density of the nuclear materials and condensation of the chromatin structures.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Molecular cloning and sequence analysis of the promoter region of mouse cyclin D1 gene: implication in phorbol ester-induced tumour promotion

Cyclin D1 is a cell cycle regulatory protein, which acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery, particularly during G1 phase of the cell cycle. Previous study using promotion-sensitive JB6 mouse epidermal cells, an in vitro model of the promotion stage of multistage carcinogenesis, showed that the expression of cyclin D1 is stimulated in the presence (but not in the absence) of 12-O-tetradecanoylphorbol-13-acetate (TPA) in these cells maintained under anchorage-independent culture conditions. In the present study, to explore the molecular basis of this observation, the promoter region of mouse cyclin D1 gene was cloned and sequenced (GenBank accession number AF212040). Dot matrix comparison of mouse, human and rat promoter sequences indicated that the mouse promoter is homologous to the human and more so to the rat promoters. The mouse promoter, like human and rat promoters, lacks canonical TATA-box or TATA-like sequence, but it has one or possibly two initiator (Inr) or Inr-like sequences. Energy dot plot analysis predicted that the mouse promoter consists of three domains: (1) the 3' domain contains NF-kappaB response element, cAMP-response element (CRE), Inr or Inr-like elements, Sp1 binding site and Oct 1 (2) the middle domain contains another Sp1 binding site, E-box and E2F binding site and (3) the 5' domain contains TPA-response element (TRE) and a tandem silencer element. The cyclin D1 promoter sequence of either promotion-sensitive or resistant JB6 mouse epidermal cells was, except for a few minor differences, essentially identical to the sequence determined for a mouse genomic clone. Since TPA is capable of stimulating the expression of cyclin D1 not only through TRE but also through CRE and NF-kappaB response element in the promoter, we tentatively propose a sequence of events that possibly leads to TPA-induced, anchorage-independent synthesis of cyclins D1 and A in the promotion-sensitive JB6 mouse epidermal cells.     

Synthesis of alpha and gamma interferons by a human cutaneous lymphoma with helper T-cell phenotype

A cloned human cutaneous lymphoma Hut102-B2 with helper T-cell phenotype (Leu1⁺, Leu2a^?, Leu3a⁺) was found to produce substantial quantities of interferon (IFN) on induction with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Whereas only trace amounts of IFN were secreted by Hut102-B2 cells spontaneously, up to 8000 laboratory units/ ml of IFN were synthesized under the optimal conditions of TPA induction. Characterization studies including neutralization by specific antisera to IFNs and determination of the activities in human and bovine cells disclosed that the IFN produced by Hut102-B2 cells exposed to TPA was a mixture of immune IFN (IFN-γ) and leukocyte IFN (IFN-α) made in approximately equal amounts in terms of antiviral activity.     

Yeast phosphofructokinase. 3. Comparison of the allosteric properties of PFKs and PFKd(MgF + )

Yeast phosphofructokinase (PFK) exists in two forms, an ATP-sensitive form, PFKs, and a desensitized form, PFKd(MgF⁺). PFKs exhibits sigmoidal kinetics with respect to Fru-6-P, whereby the S_{0.5, Fru-6-P} is determined by [ATP]. This form of PFK is inhibited by ATP and citrate and allosterically activated by Fru-6-P and AMP. NH₄⁺ activates PFKs and enhances its affinity for substrate Fru-6-P (1–3).PFKd(MgF⁺) in contrast is not inhibited by ATP and citrate, nor activated by Fru-6-P and AMP. Kinetics of the reaction with PFKd(MgF⁺) with respect to Fru6-P are hyperbolic, with $\text{K}{}_{\mathrm{m}}\text{=}\text{1}\text{4}\text{?}\text{1}\text{5}$ of S_{0.5, Fm-6-P} for PFKs. NH₄⁺ strongly activates this form.In terms of the model of Monod et al. (4), PFKd(MgF⁺) corresponds to a fixed R-conformation, while PFKs is a limiting T-conformation.     

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells

Phospholipase A₂ activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A₂ activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-³H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7–14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E₁ did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A₂ activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A₂, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A₂ activity was also related to previously found differences in the ability of the clones to develop desensitization to β-adrenergic hormones or prostaglandin E₁.     

The Effects of Various Ions on Resting and Spike Potentials of Barnacle Muscle Fibers

Effects of monovalent cations and some anions on the electrical properties of the barnacle muscle fiber membrane were studied when the intra- or extracellular concentrations of those ions were altered by longitudinal intra-cellular injection. The resting potential of the normal fiber decreases linearly with increase of logarithm of [K⁺]_out and the decrement for a tenfold increase in [K⁺]_out is 58 mv when the product, [K⁺]_out ·[Cl^-]_out, is kept constant. It also decreases with decreasing [K⁺]_in but is always less than expected theoretically. The deviation becomes larger as [K⁺]_in increases and the resting potential finally starts to decrease with increasing [K⁺]_in for [K⁺]_in > 250 mM. When the internal K⁺ concentration is decreased the overshoot of the spike potential increases and the time course of the spike potential becomes more prolonged. In substituting for the internal K⁺, Na⁺ and sucrose affect the resting and spike potentials similarly. Some organic cations (guanidine, choline, tris, and TMA) behave like sucrose while some other organic cations (TEA, TPA, and TBA) have a specific effect and prolong the spike potential if they are applied intracellularly or extracellularly. In all cases the active membrane potential increases linearly with the logarithm of [Ca⁺⁺]_out/[K⁺]_in and the increment is about 29 mv for tenfold increase in this ratio. The fiber membrane is permeable to Cl^- and other smaller anions (Br^- and I^-) but not to acetate^- and larger anions (citrate^-, sulfate^-, and methanesulfonate^-).     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

Sodium, Potassium, and Chloride Fluxes in Intercostal Muscle from Normal Goats and Goats with Hereditary Myotonia

In isolated bundles of external intercostal muscle from normal goats and goats with hereditary myotonia the following were determined: concentrations and unidirectional fluxes of Na⁺, K⁺, and Cl^-, extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na⁺ or Cl^-, fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl^- concentration in both groups of preparations was 4 to 7 times that expected if Cl^- were distributed passively between intracellular and extracellular water. The membrane permeability to K (P_K) calculated from efflux data was (a) at 38°C, 0.365 x 10^-6 cm sec^-1 for normal and 0.492 x 10^-6 for myotonic muscle, and (b) at 25°C, 0.219 x 10^-6 for normal and 0.199 x 10^-6 for myotonic muscle. From Cl^- washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP_cl could be calculated; it was 0.413 x 10^-6 cm sec^-1 for myotonic fibers and was 0.815 x 10^-6 cm sec^-1 for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.     

Molecular principles of antigenic variation in Neisseria gonorrhoeae

The genome of Neisseria gonorrhoeae harbours many gene loci for the production of variant pili. Strain MS11 has two expression genes (pilE) with promoter and complete coding sequences. The remaining genes are silent (pilS) lacking the promoter and the conservative amino terminals coding sequences of pilin. The pilus genes consist of six variable minicassettes (mc's), that are flancked by strictly conserved sequences. Upon phase (P⁺ to P⁺) and antigenic (P⁺ to P^–, or vice versa) transitions minicassettes from silent loci are transferred from silent pilus gene copies to the expression gene by gene conversion. P^– variants resulting from such rearrangements still produce pilin mRNA as well as pilin, but only a few are found on the surface of those gonococci.     

Regulation of ribulose-1,5-bisphosphate carboxylase from tobacco: changes in pH response and affinity for CO2 and Mg2+ induced by chloroplast intermediates

Ribulose-1,5-bisphosphate caryboxylase-oxygenase is activated by CO₂ and Mg²⁺ in a process distinct from catalysis. The effect of chloroplast metabolites as they separately influenced either activation or catalysis of tobacco carboxylase was examined. Of the 28 metabolites examined, 13 effected activation of the carboxylase. The strongest positive effectors were NADPH, gluconate-6-P, glycerate-2-P, and glycerate-3-P. Negative effectors included ribose-5-P, fructose-6-P, glucose-6-P, and pyrophosphate. The concentration of CO₂ or Mg²⁺ necessary to produce half-maximal activation is defined as K_act. NADPH and gluconate-6-P decreased the K_act(CO₂) from 43 to 7.4 and 3.5 μm, respectively (pH 8.0, 5 mm MgCl₂). They also decreased the K_act(M.g²⁺), but had little affect on the affinity of the enzyme for CO₂ during the catalytic process. Increasing Mg²⁺ concentration decreased the K_act(CO₂) and increasing CO₂ concentration decreased the K_act-(Mg²⁺). NADP⁺ and gluconate-6-P also affected the pH profile of activation, shifting it toward lower pH values. Changes in activation had no effect on the pH profile for catalysis of CO₂ fixation. Effectors influenced ribulose-1,5-bisphosphate oxygenase in a manner analogous to the carboxylase. At air levels of O₂ and CO₂, the ratio of carboxylase to oxygenase activity was not changed by the presence of effectors, including hydroxylamine.