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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Rational Control of Off‐State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement**

Abstract Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non‐fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off ‐state conformations, trans1 and trans2 , located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off ‐state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high‐level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue‐ and red‐shifts of the absorption bands associated to trans1 and trans2 , respectively. Thus, due to elimination of trans2 , the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two‐fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2‐V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2003

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Ciszak, Ewa↗
NASA NTRS1986

Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

NASA Discipline Musculoskeletal↗
DOE OSTI2023

Aim18p and Aim46p are chalcone isomerase domain–containing mitochondrial hemoproteins in Saccharomyces cerevisiae

Chalcone isomerases (CHIs) have well-established roles in the biosynthesis of plant flavonoid metabolites. Saccharomyces cerevisiae possesses two predicted CHI-like proteins, Aim18p (encoded by YHR198C) and Aim46p (YHR199C), but it lacks other enzymes of the flavonoid pathway, suggesting that Aim18p and Aim46p employ the CHI fold for distinct purposes. Here, we demonstrate using proteinase K protection assays, sodium carbonate extractions, and crystallography that Aim18p and Aim46p reside on the mitochondrial inner membrane and adopt CHI folds, but they lack select active site residues and possess an extra fungal-specific loop. Consistent with these differences, Aim18p and Aim46p lack CHI activity and also the fatty acid–binding capabilities of other CHI-like proteins, but instead bind heme. We further show that diverse fungal homologs also bind heme and that Aim18p and Aim46p possess structural homology to a bacterial hemoprotein. Collectively, our work reveals a distinct function and cellular localization for two CHI-like proteins, introduces a new variation of a hemoprotein fold, and suggests that ancestral CHI-like proteins were hemoproteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex

Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1–EXT2 (EXT1–2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold β1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. Furthermore, the two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Diffraction-quality, ultraflexible protein single crystals engineered with DNA

DNA-functionalized colloidal nanoparticles assemble through flexible, nanoscale DNA hybridization interactions that limit atomic-level structural order. Here, we report a valence-centric strategy that enables DNA-bonded, protein single crystals with unconventional mechanical properties. An octameric enzyme, glutarate L-2-hydroxylase, was site- and number-selectively conjugated with eight self-complementary single-stranded DNA, yielding octavalent molecular bonds. The resulting conjugate assembled into the designed body-centered tetragonal crystals that diffracted to 1.42- to 2.61-angstrom resolution, with contacts mediated by B-form DNA helices spanning 17 to 25 angstroms. Increasing oligonucleotide length induces anisotropic lattice expansion while preserving atomic periodicity, even with partial DNA occupancy. Mechanistic studies suggest that the dynamic motion of unhybridized DNA facilitates crystallization, analogous to fluctuating electron clouds in atomic bonding. Compared with native protein crystals, DNA-hybridized crystals are 23-fold softer. These results challenge the assumption that flexibility is incompatible with structural order and establish a programmable framework for biomolecular crystallization and nanomaterials engineering with atomic precision.

Han, Zhenyu [Department of Chemistry, Northwestern↗
DOE OSTI2023

Pseudomonas aeruginosa Dps (PA0962) Functions in H 2 O 2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe 2+ utilizing H 2 O 2 (not O 2 ) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H 2 O 2 -mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H 2 O 2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe 2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H 2 O 2 or O 2 but requires divalent cations and 12-mer Pa Dps.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

NASA Discipline Cell Biology↗
NASA NTRS2022

Design of synthetic collagens that assemble into supramolecular banded fibers as a functional biomaterial testbed

Collagens are the most abundant proteins of the extracellular matrix, and the hierarchical folding and supramolecular assembly of collagens into banded fibers is essential for mediating cell-matrix interactions and tissue mechanics. Collagen extracted from animal tissues is a valuable commodity, but suffers from safety and purity issues, limiting its biomaterials applications. Synthetic collagen biomaterials could address these issues, but their construction requires molecular-level control of folding and supramolecular assembly into ordered banded fibers, comparable to those of natural collagens. Here, we show an innovative class of banded fiber-forming synthetic collagens that recapitulate the morphology and some biological properties of natural collagens. The synthetic collagens comprise a functional-driver module that is flanked by adhesive modules that effectively promote their supramolecular assembly. Multiscale simulations support a plausible molecular-level mechanism of supramolecular assembly, allowing precise design of banded fiber morphology. We also experimentally demonstrate that synthetic fibers stimulate osteoblast differentiation at levels comparable to natural collagen. This work thus deepens understanding of collagen biology and disease by providing a ready source of safe, functional biomaterials that bridge the current gap between the simplicity of peptide biophysical models and the complexity of in vivo animal systems.

Jinyuan Hu↗
NASA NTRS1972

Evolution of ribonuclease in relation to polypeptide folding mechanisms.

Comparisons of the N-terminal region of pancreatic RNAase in seven species are presented, taking into account cow, bison, deer, rat, pig, kangaroo, and turtle. The available limited evidence on hypervariable regions indicates that there is still an evolutionary constraint on them. It is proposed that there is a selection pressure acting on all regions of a protein sequence in evolution. Mutations that tend to obstruct the folding process can lead to various intensities of selection pressure.

Barnard, E. A.↗
DOE OSTI2025

Plasma proteomic biomarkers of physical frailty in heart failure: a propensity score matched discovery-based pilot study

Background: Physical frailty is highly prevalent in heart failure (HF), but we lack an understanding of the underlying pathophysiology. Proteomics evaluation of plasma samples may elucidate potential mechanisms and biomarkers of physical frailty in HF. We aimed to identify plasma proteomic biomarkers that are differentially expressed between physically frail and non physically frail adults with HF. Methods: This was a secondary analysis of a subset of data and plasma samples from a study of frailty among patients with New York Heart Association (NYHA) Functional Classification I-IV HF. Physical frailty was measured using the Frailty Phenotype Criteria. Propensity score matching was used to match pairs of physically frail (n = 20) vs. non-physically frail (n = 20) patients on clinical characteristics. Plasma samples were processed using a sensitive liquid chromatography mass spectrometry platform, utilizing a multiplexed tandem mass tag-labeled quantitative proteomics approach. Differentially expressed proteins were quantified individually using paired t tests with associated log fold change of 0.3 and Fisher’s combined p values. Results: The sample (n = 40) was 62.8±16.9 years old, 58% female, and 55% NYHA Class III/IV. Proteomics analysis revealed 7 proteins differentially expressed using full differential criteria: matrix metalloproteinase-14 was downregulated in frailty, and copine-1, low affinity immunoglobulin gamma Fc region receptor III-A and III-B, probable non-functional immunoglobulin kappa variable 2D-24, glutathione S-transferase Mu 1, and argininosuccinate lyase were upregulated in frailty. Conclusions: Proteomic biomarkers related to the immune system, stress response, and detoxification were differentially expressed between physically frail and non-physically frail adults with HF.

Biomarkers↗
NASA NTRS1996

Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

Takezawa, D.↗
NASA NTRS1991

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

NASA Discipline Number 29-20↗
DOE OSTI2026

Enhancing Sensitivity in Targeted Single-Cell Proteomics by Coupling a Dual Ion Funnel Interface with Triple Quadrupole Mass Spectrometer

Single-cell proteomics (SCP) has emerged as a powerful approach for understanding cellular heterogeneity and biological processes at unprecedented resolution. However, the extremely limited protein content of individual cells (femtogram to picogram levels) pushes current mass spectrometry instrumentation to its sensitivity limits, creating a critical analytical bottleneck. While selected reaction monitoring (SRM) using triple quadrupole (QqQ) instruments 1 offers advantages in sensitivity and reproducibility for targeted proteomics quantification, SRM still struggles with sensitivity for quantification of moderate- or low-abundance proteins from single-cell sample amounts. Here, we report the development and systematic evaluation of a dual ion funnel interface designed to address the sensitivity limitation by significantly enhancing ion transmission efficiency in commercial QqQ mass spectrometers. The dual ion funnel interface, composed of a curved S-funnel followed by a conventional ion funnel, improves ion transmission efficiency while reducing chemical noise through selective ion focusing. The performance of the dual ion funnel interface was systematically compared to standard interface on a TSQ Vantage platform across samples with different levels of complexity. The dual funnel interface demonstrated to provide up to 25-fold improvement in sensitivity across a wide range of protein concentrations in different biological matrices (low complex mouse macrophage and high complex human cells). Critically, enhanced sensitivity was accompanied by increased analytical reproducibility with lower coefficient of variations. Most importantly, the dual funnel interface enabled reliable quantification of low-abundance proteins that were barely detectable or not detected by the standard interface, extending analysis to single-cell equivalent amounts while maintaining excellent reproducibility. These results demonstrate that the dual funnel interface addresses the critical bottleneck in quantitative targeted proteomics, providing a technological foundation for ultrasensitive targeted SCP that requires both high sensitivity and robust quantitative performance.

Min, Sehong↗
NASA NTRS1987

Geometrical analysis of Cys-Cys bridges in proteins and their prediction from incomplete structural information

Analysis of C-alpha atom positions from cysteines involved in disulphide bridges in protein crystals shows that their geometric characteristics are unique with respect to other Cys-Cys, non-bridging pairs. They may be used for predicting disulphide connections in incompletely determined protein structures, such as low resolution crystallography or theoretical folding experiments. The basic unit for analysis and prediction is the 3 x 3 distance matrix for Cx positions of residues (i - 1), Cys(i), (i +1) with (j - 1), Cys(j), (j + 1). In each of its columns, row and diagonal vector--outer distances are larger than the central distance. This analysis is compared with some analytical models.

NASA Discipline Exobiology↗
DOE OSTI2022

Structures of the TMC-1 complex illuminate mechanosensory transduction

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid–protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Structure, Function, Self-Assembly and Origin of Simple Membrane Proteins

Integral membrane proteins perform such essential cellular functions as transport of ions, nutrients and waste products across cell walls, transduction of environmental signals, regulation of cell fusion, recognition of other cells, energy capture and its conversion into high-energy compounds. In fact, 30-40% of genes in modem organisms codes for membrane proteins. Although contemporary membrane proteins or their functional assemblies can be quite complex, their transmembrane fragments are usually remarkably simple. The most common structural motif for these fragments is a bundle of alpha-helices, but occasionally it could be a beta-barrel. In a series of molecular dynamics computer simulations we investigated self-organizing properties of simple membrane proteins based on these structural motifs. Specifically, we studied folding and insertion into membranes of short, nonpolar or amphiphatic peptides. We also investigated glycophorin A, a peptide that forms sequence-specific dimers, and a transmembrane aggregate of four identical alpha-helices that forms an efficient and selective voltage-gated proton channel was investigated. Many peptides are attracted to water-membrane interfaces. Once at the interface, nonpolar peptides spontaneously fold to a-helices. Whenever the sequence permits, peptides that contain both polar and nonpolar amino also adopt helical structures, in which polar and nonpolar amino acid side chains are immersed in water and membrane, respectively. Specific identity of side chains is less important. Helical peptides at the interface could insert into the membrane and adopt a transmembrane conformation. However, insertion of a single helix is unfavorable because polar groups in the peptide become completely dehydrated upon insertion. The unfavorable free energy of insertion can be regained by spontaneous association of peptides in the membrane. The first step in this process is the formation of dimers, although the most common are aggregates of 4-7 helices. The helices could arrange themselves such that they formed pores capable of transporting ions and small molecules across membranes. Stability of transmembrane aggregates of simple proteins is often only marginal and, therefore, it can be regulated by environmental signals or small sequence modifications in the region of interhelical interactions. A key step in the earliest evolution of membrane proteins was the emergence of selectivity for specific substrates. Many channels could become selective if one or only a few properly chosen amino acids are properly placed along the channel, acting as filters or gates. This is a convenient evolutionary solution because it does not require imposing conditions on the whole sequence.

Pohorille, Andrew↗
NASA NTRS1988

Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

NASA Program Space Biology↗