Effect of varying thickness properties of the slow release fertilizer films on morphology, biodegradability, urea release, soil health, and plant growth.

Green biomass is a renewable and biodegradable material that has the potential use to trap urea to develop a high-efficiency urea fertilizer for crops' better performance. Current work examined the morphology, chemical composition, biodegradability, urea release, soil health, and plant growth effects of the SRF films subjected to changes in the thickness of 0.27, 0.54, and 1.03 mm. The morphology was examined by Scanning Electron Microscopy, chemical composition was analyzed by Infrared Spectroscopy, and biodegradability was assessed through evolved CO2 and CH4 quantified through Gas Chromatography. The chloroform fumigation technique was used for microbial growth assessment in the soil. The soil pH and redox potential were also measured using a specific probe. CHNS analyzer was used to calculate the total carbon and total nitrogen of the soil. A plant growth experiment was conducted on the Wheat plant (Triticum sativum). The thinner the films, the more they supported the growth and penetration of the soil's microorganisms mainly the species of fungus possibly due to the presence of lignin in films. The fingerprint regions of the infrared spectrum of SRF films showed all films in soil changed in their chemical composition due to biodegradation but the increase in the thickness possibly provides resistance to the films' losses. The higher thickness of the film delayed the rate and time for biodegradation and the release of methane gas in the soil. The 1.03 mm film (47% in 56 days) and 0.54 mm film (35% in 91 days) showed the slowest biodegradability as compared to the 0.27 mm film with the highest losses (60% in 35 days). The slow urea release is more affected by the increase in thickness. The Korsymer Pappas model with release exponent value of < 0.5 explained the release from the SRF films followed the quasi-fickian diffusion and also reduced the diffusion coefficient for urea. An increase in the pH and decrease in the redox potential of the soil is correlated with higher total organic content and total nitrogen in the soil in response to amending SRF films with variable thickness. Growth of the wheat plant showed the highest average plant length, leaf area index and grain per plant in response to the increase in the film's thickness. This work developed an important knowledge to enhance the efficiency of film encapsulated urea that can better slow the urea release if the thickness is optimized.

Diclofenac derivatives as concomitant inhibitors of cholinesterase, monoamine oxidase, cyclooxygenase-2 and 5-lipoxygenase for the treatment of Alzheimer's disease: synthesis, pharmacology, toxicity and docking studies.

Targeting concomitantly cholinesterase (ChEs) and monoamine oxidases (MAO-A and MAO-B) is a key strategy to treat multifactorial Alzheimer's disease (AD). Moreover, it is reported that the expression of cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) is increased significantly in the brain of AD patients. Using the triazole of diclofenac 12 as a lead compound, we synthesized a variety of analogs as multipotent inhibitors concomitantly targeting COX-2, 5-LOX, AChE, BChE, MAO-A and MAO-B. A number of compounds showed excellent in vitro inhibition of the target biological macromolecules in nanomolar concentration. Compound 39 emerged as the most potent multitarget ligand with IC50 values of 0.03 µM, 0.91 µM, 0.61 µM, 0.01 µM 0.60 µM and 0.98 µM towards AChE, BChE, MAO-A, MAO-B, COX-2 and 5-LOX respectively. All the biologically active compounds were found to be non-neurotoxic and blood-brain barrier penetrant by using PAMPA assay. In a reversibility assay, all the studied active compounds showed reversibility and thus were found to be devoid of side effects. MTT assay results on neuroblastoma SH-SY5Y cells showed that the tested compounds were non-neurotoxic. An in vivo acute toxicity study showed the safety of the synthesized compounds up to a 2000 mg kg-1 dose. In docking studies three-dimensional construction and interaction with key residues of all the studied biological macromolecules helped us to explain the experimental results.

Impaired calcium signalling and neuropsychiatric disorders in Darier disease: An exploratory review.

Darier (Darier-White) disease (DD) is an autosomal dominant skin disorder caused by pathogenic mutations in the ATP2A2 gene which encodes a calcium ATPase in the sarco-endoplasmic reticulum (SERCA2). Defects in the SERCA2 protein lead to an impairment of cellular calcium homeostasis, which in turn, triggers cell death pathways. There is a high prevalence of neuropsychiatric disorders in patients affected by this condition, namely intellectual disability, bipolar disorder, schizophrenia, and suicidality. Though these associations have been well-documented over the years, little has been discussed or investigated regarding the pathophysiological mechanisms. The goal of this article is to review the literature related to the most commonly associated neuropsychiatric disorders found in patients with DD, highlight the pathophysiological mechanisms underlying each condition, and examine potential interventions that may be of interest for future development. A literature search was performed using PubMed to access and review relevant articles published in the last 40 years. Keywords searched included Darier disease neuropsychiatric, Darier disease pathophysiology, SERCA2 central nervous system, SERCA 2 skin, ATP2A2 central nervous system, ATP2A2 skin, sphingosine-1-phosphate signalling skin, sphingosine-1-phosphate signalling central nervous system, P2X7 receptor skin, and P2X7 receptor central nervous system. Our search resulted in 2692 articles, of which 61 articles were ultimately included in this review.

It Is Time for an Oil Change: Polyunsaturated Fatty Acids and Human Health.

Polyunsaturated fatty acids (PUFAs) such as docosahexaneoic acid (DHA) and eicosapentaneoic acid (EPA), play a critical role in a variety of neuronal functions, including facilitating neuronal growth and differentiation, increasing the density of the neuritic network, modulating cell membrane fluidity, regulating intracellular signaling and gene expression, and exhibiting antioxidant characteristics. Dietary DHA is selectively enriched and actively retained in the central nervous system, mainly in synaptic membranes, dendrites, and photoreceptors. In this review, we highlight the myriad roles of PUFAs in brain function and human health. Diets rich in DHA are inversely proportional to cognitive decline and incidence of neurodegenerative disorders. Conversely, diets deficient in DHA impair the proper development of brain and the visual system in children and increase risk of brain disorders in the elderly. Finally, DHA and EPA have been shown to reduce inflammation and may prove to be beneficial in reducing the severity of the SARS-COVID infection.

Enzyme inhibition and antibacterial potential of 4-Hydroxycoumarin derivatives

The 4-Hydroxycoumarin derivatives are known to show a broad spectrum of pharmacological applications. In this paper we are reporting the synthesis of a new series of 4-Hydroxycoumarin derivatives synthesized through Knovenegal condensation; they were characterized by using UV-Vis, FT-IR, NMR spectroscopies. The synthesized compounds were evaluated for antibacterial activity against Staphylococcus aureus and Salmonella typhimurium strains. The compounds (2), (3) and (8) showed favorable antibacterial activity with zone of inhibitions 26.5± 0.84, 26.0 ± 0.56 and 26.0 ± 0.26 against Staphylococcus aureus (Gram-positive) respectively. However, the compounds (5) and (9) were found more active with 19.5 ± 0.59 and 19.5 ± 0.32 zone of inhibitions against Salmonella typhimurium (Gram-negative). Whereas, in urease inhibition assay, none of the synthesized derivatives showed significant anti-urease activity; although, in carbonic anhydrase-II inhibition assay, the compound (2) and (6) showed enzyme inhibition activity with IC50 values 263±0.3 and 456±0.1, respectively.

The plasma membrane calcium pumps-The old and the new.

The plasma membrane Ca2+-ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca2+) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca2+ [Ca2+]i. Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca2+]i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration.

Changes in thyroid function status of suicidal patients

Abstract Background This study was carried out at Punjab Institute of Mental Health and Centre for Nuclear Medicine Mayo Hospital, Lahore. It is aimed at the possible association of thyroid malfunctioning with suicide attempts of patients. Objective Determination of thyroid function status of suicidal psychiatric patients and their comparison with psychiatric patients without suicide attempt or ideation. Methods Total 54 patients with either past history of suicide attempt or current suicidal ideation were selected for analysis of their thyroid function status (age 15-55 years). Age matched 50 non-suicide psychiatric patients were included for comparison. Results Two patients with suicide attempt had overt thyroid dysfunction. Remaining patients had serum FT4, FT3 and TSH level within normal range. Suicide attempter patients had lower FT4 but increased FT3 and TSH levels compared to suicidal ideation patients. Serum FT4 and TSH levels in suicidal patients were not different from psychiatric patients. Serum FT3 in suicidal patients was lower than psychiatric patients (3.7 ± 0.8 vs. 4.3 ± 0.5; p < 0.05). Female suicidal patients had lower FT3 levels compared to male patients (3.4 ± 0.6 vs. 3.9 ± 0.8 pmol/L; p < 0.05). Discussion Local suicidal patients have higher incidence of overt thyroid disorder and lower FT3 levels compared to non-suicidal psychiatric patients.

CHLOROGENIC ACID: A PHARMACOLOGICALLY POTENT MOLECULE.

Chlorogenic acid (CGA; (IS,3R,4R.5R)-3-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid) is a naturally occurring polyphenol mostly present in vegetables and fruits. CGA is a prominent component of Traditional Chinese Medicines and is known for various pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory and hepatoprotective etc. This mini-review is an attempt to summarize the available literature in the last decade and to point out future perspectives in this area of research.

Effects of gangliosides on the activity of the plasma membrane Ca2+-ATPase.

Control of intracellular calcium concentrations ([Ca(2+)]i) is essential for neuronal function, and the plasma membrane Ca(2+)-ATPase (PMCA) is crucial for the maintenance of low [Ca(2+)]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca(2+) homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca(2+) transporter.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

Effects of paraquat-induced oxidative stress on the neuronal plasma membrane Ca(2+)-ATPase.

Oxidative stress leads to the disruption of calcium homeostasis in brain neurons; however, the direct effects of oxidants on proteins that regulate intracellular calcium ([Ca(2+)](i)) are not known. The calmodulin (CaM)-stimulated plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in regulating [Ca(2+)](i). Our previous in vitro studies showed that PMCA present in brain synaptic membranes is readily inactivated by a variety of reactive oxygen species (ROS). The present studies were conducted to determine the vulnerability of PMCA to ROS generated in neurons as would probably occur in vivo. Primary cortical neurons were exposed to paraquat (PQ), a redox cycling agent that generates intracellular ROS. Low concentrations of PQ (5-10 microM) increased PMCA basal activity by two-fold but abolished its sensitivity to CaM. Higher concentrations (25-100 microM) inhibited both components of PMCA activity. Immunoblots showed the formation of high-molecular-weight PMCA aggregates. Additionally, PMCA showed evidence of proteolytic degradation. PMCA proteolysis was prevented by a calpain inhibitor, suggesting a role for calpain. Our findings suggest that PMCA is a sensitive target of oxidative stress in primary neurons. Inactivation of this Ca(2+) transporter under prolonged oxidative stress could alter neuronal Ca(2+) signaling.

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca2+-ATPase: dependence on enzyme and Ca2+ concentrations.

Calmodulin (CaM) is a Ca2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the Kd for the interaction of CaM with the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca2+ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K(d) in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K(d) value of CaM with PMCA (5.8 +/- 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K(d) = 9.8 +/- 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca2+ to the binding sites in CaM.

Interchange of autoinhibitory domain conformations in plasma-membrane Ca2+-ATPase-calmodulin complexes.

The Ca2+ signaling protein calmodulin (CaM) stimulates Ca2+ pumping in the plasma-membrane Ca2+-ATPase (PMCA) by binding to an autoinhibitory domain, which then dissociates from the catalytic domain of PMCA to allow full activation of the enzyme. We measured single-molecule fluorescence trajectories with polarization modulation to track the conformation of the autoinhibitory domain of PMCA pump bound to fluorescently labeled CaM. Interchange of the autoinhibitory domain between associated and dissociated conformations was detected at a physiological Ca2+ concentration of 0.15 microM, where the enzyme is only partially active, but not at 25 microM, where the enzyme is fully activated. In previous work we showed that the conformation of the autoinhibitory domain in PMCA-CaM complexes could be monitored by the extent of modulation of single-molecule fluorescence generated with rotating excitation polarization. In the present work, we determined the timescale of association and dissociation of the autoinhibitory domain with the catalytic regions of the PMCA. Association of the autoinhibitory domain was rare at a high Ca2+ concentration (25 microM). At a lower Ca2+ concentration (0.15 microM), conformations of the autoinhibitory domain interchanged with a dissociation rate of 0.042 +/- 0.011 sec(-1) and an association rate of 0.023 +/- 0.006 sec-1. The results indicate that the response time of PMCA upon a reduction in Ca2+ is limited to tens of seconds by autoinhibitory dynamics. This property may reduce the sensitivity of PMCA to transient reductions in intracellular Ca2+. We suggest that the dynamics of the autoinhibitory domain may play a novel role in regulating PMCA activity.

Partitioning of the plasma membrane Ca2+-ATPase into lipid rafts in primary neurons: effects of cholesterol depletion.

Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability.

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.

Theoretically predicted structures of plasma membrane Ca(2+)-ATPase and their susceptibilities to oxidation.

Oxidative damage to the plasma membrane Ca(2+)-ATPase (PMCA) appears to contribute to the decreased clearance of intracellular Ca(2+) in the neurons of aged brain, possibly contributing to its vulnerability to numerous age-related diseases such as Alzheimer's disease. The precise sites of oxidative susceptibility have not been identified. However, it is known that calmodulin (CaM) protects the purified PMCA against oxidative inactivation, perhaps via conformational restructuring of the protein through dissociation of a 20 residue domain (C20W) in the C-terminal region that function as a CaM-binding site. In order to postulate likely oxidation sites and the mechanism underlying the protection offered by CaM, we have generated a three-dimensional model of PMCA via a combination of homology/comparative modeling, threading, protein-protein docking, and guidance from prior biochemical and analytical studies. The resulting model was validated based on surface polarity/hydrophobicity profiling, standard ProCheck, WhatIF, and PROVE checks, as well as comparison with empirical structure-function observations. This model was then used to identify likely oxidation sites by comparing time-averaged solvent accessibility of potentially oxidizable surface residues as measured from molecular dynamics simulations of intact PMCA and the PMCA sequence from which C20W has been deleted. The resulting model complex has permitted us to identify three amino acids whose solvent accessibility is greatly reduced by the C20W dissociation: Tyr 589, Met 622, and Met 831.

Single-molecule characterization of the dynamics of calmodulin bound to oxidatively modified plasma-membrane Ca2+-ATPase.

We used single-molecule fluorescence spectroscopy to probe the conformation of calmodulin (CaM) bound to oxidatively modified plasma-membrane Ca(2+)-ATPase (PMCAox). We found that oxidative modification altered the coupling between the ATP binding domain and the autoinhibitory domain. Oxidative modification of PMCA is known to result in a loss of activity for the enzyme. Conformations of PMCAox-CaM complexes were probed by single-molecule polarization modulation spectroscopy, which measured the orientational mobility of fluorescently labeled CaM bound to PMCAox. We detected an enhanced population of PMCAox-CaM complexes with a low orientational mobility in the presence of ATP, whereas nonoxidized PMCA-CaM complexes existed almost exclusively in a high-mobility state in the presence of ATP. We have previously attributed such high-mobility states to PMCA-CaM complexes with a dissociated autoinhibitory/CaM binding domain, whereas the lower-mobility state was attributed to autoinhibited PMCA-CaM complexes with a nondissociated autoinhibitory domain [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. In the absence of ATP, the orientational mobility distributions are similar for CaM complexed with oxidized PMCA or nonoxidized PMCA. These results suggest that oxidative modification of PMCA reduced the propensity of the autoinhibitory domain to dissociate from binding sites near the catalytic core of the enzyme with bound nucleotide upon CaM stimulation in the presence of Ca(2+). This interpretation was further supported by chymotrypsin proteolysis, which probes the tightness of binding of the autoinhibitory domain to sites near the catalytic core of the enzyme. Enhanced proteolysis was observed for PMCA upon binding CaM or ATP. In contrast, proteolysis was partially blocked for oxidatively modified PMCA, even in the presence of ATP.

Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin.

We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.

Single-molecule dynamics of the calcium-dependent activation of plasma-membrane Ca2+-ATPase by calmodulin.

The plasma membrane calcium-ATPase (PMCA) helps to control cytosolic calcium levels by pumping out excess Ca2+. PMCA is regulated by the Ca2+ signaling protein calmodulin (CaM), which stimulates PMCA activity by binding to an autoinhibitory domain of PMCA. We used single-molecule polarization methods to investigate the mechanism of regulation of the PMCA by CaM fluorescently labeled with tetramethylrhodamine. The orientational mobility of PMCA-CaM complexes was determined from the extent of modulation of single-molecule fluorescence upon excitation with a rotating polarization. At a high Ca2+ concentration, the distribution of modulation depths reveals that CaM bound to PMCA is orientationally mobile, as expected for a dissociated autoinhibitory domain of PMCA. In contrast, at a reduced Ca2+ concentration a population of PMCA-CaM complexes appears with significantly reduced orientational mobility. This population can be attributed to PMCA-CaM complexes in which the autoinhibitory domain is not dissociated, and thus the PMCA is inactive. The presence of these complexes demonstrates the inadequacy of a two-state model of Ca2+ pump activation and suggests a regulatory role for the low-mobility state of the complex. When ATP is present, only the high-mobility state is detected, revealing an altered interaction between the autoinhibitory and nucleotide-binding domains.

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.