Positive Cooperativity Research Articles

Positive cooperativity in binding: Spectroscopic characterization and dissociation energy of the anisole…(CH4)2 complex

We have previously reported on the spectroscopy and binding energy of the anisole…methane complex, which exhibits a dual mode of binding that involves both CH/O and CH/π interactions. In this work, we seek to examine cooperativity in binding through a study of the isolated anisole-(methane)2 complex, using a combination of experiments that include mass-selected two-color resonant two-photon ionization spectroscopy (2CR2PI), two-color appearance potential (2CAP) measurements, and velocity mapped ion imaging (VMI) augmented with a complementary theoretical characterization. Using 2CAP and VMI, we derive the dissociation energies of the complex in ground (S0), excited (S1), and cation radical (D0) states. The experimental values from the two methods are in excellent agreement and are compared with selected theoretical values calculated using DFT and ab initio methods. The data show that the dissociation energy increases by some 10 % for the second methane relative to the first, with this trend being consistent across all three electronic states, indicating a cooperative binding effect where the initial solvation turns on binding of a second methane onto the opposite face. This lies in contrast to the aniline-(methane)2 complex, where recent studies have shown a negativity cooperativity, and these trends are examined.

Disaggregation-Activated pan-COX Imaging Agents for Human Soft tissue Sarcoma.

Cancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD-IMC-1, BD-IMC-2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD-IMC-1 showcased higher susceptibility to disaggregation compared to BD-IMC-2, consistent with their selective interaction with COX. Notably, the BD-IMC-1 revealed positive cooperativity binding to COX-2 at sub-micromolar ranges. Both probes showed significant fluorescence turn-on upon LPS or PMA triggered COX-2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos-LM2) up to 2-fold increase with negligible toxicity. More importantly, the BD-IMC-1 demonstrated their practical imaging for COX-2 positive cells in paraffin-fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Disaggregation‐Activated pan‐COX Imaging Agents for Human Soft tissue Sarcoma

AbstractCancer stem cells are pivotal players in tumors initiation, growth, and metastasis. While several markers have been identified, there remain challenges particularly in heterogeneous malignancies like adult soft tissue sarcomas, where conventional markers are inherently overexpressed. Here, we designed BODIPY scaffold fluorescence probes (BD‐IMC‐1, BD‐IMC‐2) that activate via disaggregation targeting for cyclooxygenase (COX), a potential marker for CSCs in sarcoma in clinical pathology. Based on their structures, BD‐IMC‐1 showcased higher susceptibility to disaggregation compared to BD‐IMC‐2, consistent with their selective interaction with COX. Notably, the BD‐IMC‐1 revealed positive cooperativity binding to COX‐2 at sub‐micromolar ranges. Both probes showed significant fluorescence turn‐on upon LPS or PMA triggered COX‐2 upregulation in live RAW264.7, HeLa, and human sarcoma cell line (Saos‐LM2) up to 2‐fold increase with negligible toxicity. More importantly, the BD‐IMC‐1 demonstrated their practical imaging for COX‐2 positive cells in paraffin‐fixed human sarcoma tissue. Considering the fixed tissues are most practiced pathological sample, our finding suggests a potential of disaggregation activated chemosensor for clinical applications.

Dynamic Structures and Fast Transition Kinetics of Oxidized G-Quadruplexes.

8-oxoguanines (8-oxoG) in cells form compromised G-quadruplexes (GQs), which may vary GQ mediated gene regulations. By mimicking molecularly crowded cellular environment using 40% DMSO or sucrose, here it is found that oxidized human telomeric GQs have stabilities close to the wild-type (WT) GQs. Surprisingly, while WT GQs show negative formation cooperativity between a Pt(II) binder and molecularly crowded environment, positive cooperativity is observed for oxidized GQ formation. Single-molecule mechanical unfolding reveals that 8-oxoG sequence formed more diverse and flexible structures with faster folding/unfolding transition kinetics, which facilitates the Pt(II) ligand to bind the best-fit structures with positive cooperativity. These findings offer new understanding on structures and properties of oxidized G-rich species in crowded environments. They also provide insights into the design of better ligands to target oxidized G-rich structures formed under oxidative cell stress.

Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

Revealing the Origin of Cooperative Adsorption of Chains on Nanoparticle Surfaces through Coarse-Grained Simulations.

This work aims to deepen our understanding of the molecular origin of the recently observed phenomenon of polymer cooperative adsorption onto faceted nanoparticle (NP) surfaces. By exploring a large parameter space for polymer/NP interactions through coarse-grained (CG) molecular dynamics (MD) simulations, it is found that consistent with experiments the presence or absence of cooperativity is related to solvent quality and relative interaction strengths between the polymer and the adsorbent. Specifically, positive cooperativity is associated with stronger polymer-polymer interaction than polymer-surface interactions and vice versa for negative cooperativity. This contrast in interaction energies manifests in positive cooperativity (i.e., increased affinity) and negative cooperativity (i.e., decreased affinity) as concentration increases. It is also found that increasing chain length strengthens cooperativity effects and that the nanoscale confinement of polymer chains to the adsorbing facet (due to weaker affinity to corners and edges) enhances positive cooperativity but weakens negative cooperativity. Moreover, adsorption onto a spherical NP shows stronger positive cooperativity but weaker negative cooperativity compared with adsorption onto a cubic NP of equal surface area. It was further found that as polymer bulk concentration increases, the free energy of adsorption decreases in positive cooperativity, increases in negative cooperativity, and is independent of concentration in noncooperative systems consistent with the phenomenological explanation of cooperativity. We further found that positive cooperativity is associated with growing fluctuations in the adsorption density at critical bulk polymer concentrations. This behavior can be attributed to the competition between enthalpic gains and entropic losses upon adsorption. Overall, our results shed light on the microscopic origin of cooperative adsorption and the role of solvent quality, which can be leveraged in, for example, controlling NP growth into target shapes and designing NP catalysts with improved performance.

Cooperative nucleic acid binding by Poly ADP-ribose polymerase 1

Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1’s normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20 and U20 oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20 and U20 oligonucleotides show that PARP’s binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1’s ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

Film-straw dual mulching improves soil fertility and maize yield in dryland farming by increasing straw-degrading bacterial abundance and their positive cooperation

Film-straw dual mulching (FMSS) has been utilized successfully for corn production in dryland farming. However, the mechanisms by which FMSS improves soil fertility and maize yields remain obscure. Therefore, a three-year field trial with four treatments, including conventional tillage (blank, BL), FM with standard fertilization (FMSF), and with straw incorporation (FMSS), was conducted between 2018 and 2020. The results demonstrated that FMSS significantly increased the concentrations of soil organic carbon (SOC, 17.48%) and total and available nitrogen and phosphorus (TN, 34.48%; TP, 16.90%; AN, 23.34% and AP, 40.95%) in the 040 cm soil profile, resulting in a 31.0% increase in maize yield. FMSS had no significant effect on soil bacterial diversity; however, it selectively increased the relative abundances of Firmicutes, Gemmatimonadetes, and Acidobacteria but decreased those of Proteobacteria, Actinobacteria, Bacteroidetes, and Nitrospirae, and dynamically regulated bacterial life strategies (mainly from k-strategies to r-strategies), thereby forming an efficient straw-degrading functional community. These three straw-degrading bacteria demonstrated a cooperative relationship, and increased labile and recalcitrant straw carbon (C) and nitrogen (N) decomposition rate, nutrient release, and soil fertility. FMSS improved the straw-degrading bacteria abundance, regulated their life strategies, promoted positive cooperation that increases straw decomposition, and increased SOC, TN, TP, AN, and AP, thereby significantly improving soil fertility and maize yield. Therefore, FMSS is an efficient management practice that can replace FM and increase maize yields in dryland farming.

Profiling the Interaction between Human Serum Albumin and Clinically Relevant HIV Reverse Transcriptase Inhibitors.

Tenofovir (TFV) is the active form of the prodrugs tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), both clinically prescribed as HIV reverse transcriptase inhibitors. The biophysical interactions between these compounds and human serum albumin (HSA), the primary carrier of exogenous compounds in the human bloodstream, have not yet been thoroughly characterized. Thus, the present study reports the interaction profile between HSA and TFV, TDF, and TAF via UV-Vis, steady-state, and time-resolved fluorescence techniques combined with isothermal titration calorimetry (ITC) and in silico calculations. A spontaneous interaction in the ground state, which does not perturb the microenvironment close to the Trp-214 residue, is classified as weak. In the case of HSA/TFV and HSA/TDF, the binding is both enthalpically and entropically driven, while for HSA/TAF, the binding is only entropically dominated. The binding constant (Ka) and thermodynamic parameters obtained via ITC assays agree with those obtained using steady-state fluorescence quenching measurements, reinforcing the reliability of the data. The small internal cavity known as site I is probably the main binding pocket for TFV due to the low steric volume of the drug. In contrast, most external sites (II and III) can better accommodate TAF due to the high steric volume of this prodrug. The cross-docking approach corroborated experimental drug-displacement assays, indicating that the binding affinity of TFV and TAF might be impacted by the presence of different compounds bound to albumin. Overall, the weak binding capacity of albumin to TFV, TDF, and TAF is one of the main factors for the low residence time of these antiretrovirals in the human bloodstream; however, positive cooperativity for TAF and TDF was detected in the presence of some drugs, which might improve their residence time (pharmacokinetic profile).

The binding and mechanism of a positive allosteric modulator of Kv3 channels

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel’s tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.

A three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D

Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.

The effect of positive inter-group contact on cooperation: the moderating role of individualism.

The effect of intergroup contact on cooperation is well documented, but little is known about the cultural moderators of this relationship. Contributing to the literature, we examined whether cultural orientation moderates the effect of positive intergroup contact on cooperation and places emphasis on individualism. By creating a gamecooperation situation by the trust game paradigm, 322 Taiwanese youth were involved in the study and completed the positive intergroup contact scale, individual-collectivism scale, and social distance scale. (1) positive intergroup contact effectively promotes cooperative behavior; (2) Taiwanese youth who have closer social distance with mainland youth demonstrate higher levels of cooperative behavior after group interactions than larger social distance; and (3) individualism has a significant moderating role in the relationship between positive inter-group contact and cooperation. The effect of positive inter-group contact on cooperation became stronger in the less individualistic group. The effect of social distance on cooperation became stronger in the less individualistic group.Cultural orientation represented by individualism is proved to be one moderato of the intergroup contact-cooperation relationship.

Exploring energy mechanisms involved in the luminescence reduction due to Ce3+ doping via core-multishell upconversion nanoparticles highly doped with Ho3+ and Yb3+

Upconversion nanoparticles (UCNPs), particularly those that are highly doped, possess outstanding properties and have demonstrated tremendous application potential. However, novel fine-tuning strategies are urgently required to enhance the upconversion luminescence (UCL) performance of UCNPs. In this study, a series of core–multishell UCNPs highly doped with Ho3+ and Yb3+ ions were synthesized via the oleate route. The X-ray diffraction (XRD), transmission electron microscopy (TEM), and UCL spectra and decay times, and pump power dependency of the synthesized UCNPs were used to examine their morphology, size, and the UCL properties. Ce3+ ions were introduced to enhance Ho3+-based red UCL, and their doping strategy was also optimized based on summarized UCL trends and detailed UCL mechanism analysis. NaHoF4:Gd3+@NaGdF4:Yb3+,Ce3+@NaGdF4:Yb3+ UCNPs that had 10 mol% Ce3+ dopants in their middle shells exhibited better UCL properties than the larger traditional NaGdF4:Yb3+,Ce3+,Ho3+ UCNPs, probably because of the combined influence of the energy interactions between the Ce3+ ions and surface groups, sensitizing energy enhancement by active shells, and the positive cooperation between the Ce3+ ions and highly-doped core–shell structures. In this study, the phenomenon of UCL decrease resulting from Ce3+ ion doping was explored. Using unique core–multishell structures with highly-doped activators and sensitizers placed within and outside the structures, respectively, the UCL decrease due to Ce3+ ion doping was attributed to a competition for photons. Doping the UCNPs with Ce3+ ions near the UCNP surfaces favors Ho3+-based red UCL. Our study findings can deepen the understanding of the Ho3+-based UCL mechanisms and increase the potential uses of NaHoF4 materials in photo-induced therapy.

Dynamic behavior of enzyme kinetics cooperative chemical reactions

This article uses computational mathematics to investigate the dynamics of cooperative occurrences in chemical reactions inside living organisms. We study the dynamics of complex systems using mathematical models based on ordinary differential equations, paying special attention to chemical equilibrium and reaction speed. Explanations of cooperation, non-cooperation, and positive cooperation are presented in our study. We analyze the stabilities of equilibrium points by a systematic analysis that makes use of the Jacobian matrix and the threshold parameter R0. We next extend our investigation to evaluate global stability and the probability of the model. Variations in k3 have a notable effect on substrate concentration probabilities, indicating that it plays an important role in reaction kinetics. Reducing k3 highlights the substrate's critical contribution to the system by extending its presence in the concentration. We find that different results were obtained for cooperative behavior: higher reaction rates at different binding sites are correlated with positive cooperativity, while slower reactions are induced by negative cooperativity. The Adams–Bashforth method is used to show numerical and graphical solutions with the help of MATLAB. Tables and graphs are used to further explain the effects of the parameters. This study underlines how well ordinary differential equations may represent the complicated system dynamics found in chemical reactions. It also provides elusive insights into cooperative occurrences, which develops our understanding of the phenomenon and serves as a foundation for future research.

CAMP binding to closed pacemaker ion channels is cooperative

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Dye-decolorizing peroxidase of Thermobifida halotolerance displays complex kinetics with both substrate inhibition and apparent positive cooperativity

Dye-decolorizing peroxidases (DyPs) have been intensively investigated for the purpose of industrial dye decolourization and lignin degradation. Unfortunately, the characterization of these peroxidases is hampered by their non-Michaelis-Menten kinetics, exemplified by substrate inhibition and/or positive cooperativity. Although often observed, the underlying mechanisms behind the unusual kinetics of DyPs are poorly understood. Here we studied the kinetics of the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroquinones, and anthraquinone dyes by DyP from the bacterium Thermobifida halotolerans (ThDyP) and solved its crystal structure. We also provide rate equations for different kinetic mechanisms explaining the complex kinetics of heme peroxidases. Kinetic studies along with the analysis of the structure of ThDyP suggest that the substrate inhibition is caused by the non-productive binding of ABTS to the enzyme resting state. Strong irreversible inactivation of ThDyP by H2O2 in the absence of ABTS suggests that the substrate inhibition by H2O2 may be caused by the non-productive binding of H2O2 to compound I. Positive cooperativity was observed only with the oxidation of ABTS but not with the two electron-donating substrates. Although the conventional mechanism of cooperativity cannot be excluded, we propose that the oxidation of ABTS assumes the simultaneous binding of two ABTS molecules to reduce compound I to the enzyme resting state, and this causes the apparent positive cooperativity.

Heterotypic interactions can drive selective co-condensation of prion-like low-complexity domains of FET proteins and mammalian SWI/SNF complex

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF (mSWI/SNF) complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.

Synthesis and cooperative guest binding of tetrameric porphyrin macrocycle

AbstractA new family of macrocyclic host molecules that possess two bisporphyrin clefts was synthesized. The tetrameric porphyrin macrocycle provides the two guest binding sites in which two electron‐deficient aromatic molecules are encapsulated through donor‐acceptor and π‐π stacking interactions. The tetrameric porphyrin macrocycle showed positive cooperativity, whereas the nonmacrocyclic analog did not. The preorganization concept drives positive cooperativity, which is directed by the entropy benefit to macrocyclization.

Cooperativity, Information Gain, and Energy Cost During Early LTP in Dendritic Spines.

We investigate a mutual relationship between information and energy during the early phase of LTP induction and maintenance in a large-scale system of mutually coupled dendritic spines, with discrete internal states and probabilistic dynamics, within the framework of nonequilibrium stochastic thermodynamics. In order to analyze this computationally intractable stochastic multidimensional system, we introduce a pair approximation, which allows us to reduce the spine dynamics into a lower-dimensional manageable system of closed equations. We found that the rates of information gain and energy attain their maximal values during an initial period of LTP (i.e., during stimulation), and after that, they recover to their baseline low values, as opposed to a memory trace that lasts much longer. This suggests that the learning phase is much more energy demanding than the memory phase. We show that positive correlations between neighboring spines increase both a duration of memory trace and energy cost during LTP, but the memory time per invested energy increases dramatically for very strong, positive synaptic cooperativity, suggesting a beneficial role of synaptic clustering on memory duration. In contrast, information gain after LTP is the largest for negative correlations, and energy efficiency of that information generally declines with increasing synaptic cooperativity. We also find that dendritic spines can use sparse representations for encoding long-term information, as both energetic and structural efficiencies of retained information and its lifetime exhibit maxima for low fractions of stimulated synapses during LTP. Moreover, we find that such efficiencies drop significantly with increasing the number of spines. In general, our stochastic thermodynamics approach provides a unifying framework for studying, from first principles, information encoding, and its energy cost during learning and memory in stochastic systems of interacting synapses.

A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cationsand pyridine dinucleotides.