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At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
NASA NTRS1997

Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

Non-NASA Center↗
NASA NTRS1994

Metabolic energy required for flight

This paper reviews data available from U.S. and U.S.S.R. studies on energy metabolism in the microgravity of space flight. Energy utilization and energy availability in space seem to be similar to those on Earth. However, negative nitrogen balances in space in the presence of adequate energy and protein intakes and in-flight exercise, suggest that lean body mass decreases in space. Metabolic studies during simulated (bed rest) and actual microgravity have shown changes in blood glucose, fatty acids, and insulin levels, suggesting that energy metabolism may be altered during flight. Future research should focus on the interactions of lean body mass, diet, and exercise in spaced and their roles in energy metabolism during space flight.

Lane, H. W.↗
NASA NTRS2003

The Cytoskeleton and Force Response Mechanisms

The long term aim of this project was to define the mechanisms by which cells sense and respond to the physical forces experienced at 1g and missing in microgravity. Identification and characterization of the elements of the cells force response mechanism could provide pathways and molecules to serve as targets for pharmacological intervention to mitigate the pathologic effects of microgravity. Mechanical forces experienced by the organism can be transmitted to cells through molecules that allow cells to bind to the extracellular matrix and through other types of molecules which bind cells to each other. These molecules are coupled in large complexes of proteins to structural elements such as the actin cytoskeleton that give the cell the ability to sense, resist and respond to force. Application of small forces to tissue culture cells causes local elevation of intracellular calcium through stretch activated ion channels, increased tyrosine phosphorylation and a restructuring of the actin cytoskeleton. Using collagen coated iron oxide beads and strong magnets, we can apply different levels of force to cells in culture. We have found that force application causes the cells to polymerize actin at the site of mechanical deformation and unexpectedly, to depolymerize actin across the rest of the cell. Observations of GFP- actin expressing cells demonstrate that actin accumulates at the site of deformation within the first five minutes of force application and is maintained for many tens of minutes after force is removed. Consistent with the reinforcement of the cytoskeletal structures underlying the integrin-bead interaction, force also alters the motion of bound magnetic beads. This effect is seen following the removal of the magnetic field, and is only partially ablated by actin disruption with cytochalsin B. While actin is polymerizing locally at the site of force application, force also stimulates a global reduction in actin filament content within the cells. We have examined the roles of several actin filament disassembly factors in the global reduction of cellular actin filaments. The calcium regulated actin filament severing protein gelsolin is not necessary for the increased actin turnover, as cells derived from gelsolin null and wildtype mice still show a reduction in total actin filament content. Instead, our work suggests that the actin binding protein cofilin may be important for these changes in actin dynamics. Cofilin binds to and enhances the disassembly of actin filaments. Using immunological methods, we observe transient changes in the phosphorylation state of cofilin upon force application that suggests that cofilin may mediate actin filament turnover. Early after force application, cofilin is transiently dephosphorylated, activating its actin disassembly activity. Subsequently, we find a hyper-phosphorylation of cofilin, rendering it inactive. This reduction in cofilin activity may explain the stability of the force induced actin structuttes. In testing this hypothesis, we aimed to generate cells that express the constituitively active kinase (LIM-kinase) that phosphorylates cofilin. lnidial attempts in the cell lines used for the our previous studies proved unsuccessful. While we prepare this work for pubication, we are continuing to study other cell lines and tissue sources to determine whether they show a reduction in F-actin content after force application.

Allen, Philip Goodwin↗
NASA NTRS1998

Identification of sucrose synthase as an actin-binding protein

Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

Non-NASA Center↗
NASA NTRS1998

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Non-NASA Center↗
NASA NTRS2016

In-Flight Personalized Medication Management

Current medication selection for treatment of astronauts during spaceflight missions is primarily dictated by the task of efficiently treating the widest possible range of physiological conditions and illnesses with a limited set of medications. Dosage and recommendations on the combination of drugs are based on the assumption of genetically equal drug sensitivity and unchanged metabolism. To our knowledge, there was no pre-flight drug sensitivity testing on a genetic level for any of the previous manned NASA space missions. Although many of the common, binary drug-drug interactions are, most likely, already considered in the ISS Medical kit composition, multi-drug and multi-drug-gene factors are not incorporated in the medication selection or prescription. Furthermore, due to the physiological changes occurring in microgravity environments, astronauts might be susceptible to potential increased drug toxicity as a result of decreased clearance of numerous drugs. In particular, perturbation of CYP450 enzymes which contribute to the hepatic metabolism of the majority of drugs may have significant effects on therapeutic efficacy and increase treatment-related toxicity5. The genes encoding the CYP450 enzymes are highly variable in humans. Inheritable variations of CYP450 hepatic metabolizer enzymes and transport proteins play a crucial role in the inter-individual variability of drug efficiency and risks of adverse drug reactions5. Additionally, there are some reports that document changes in the levels of production of drug-metabolizing enzymes in microgravity. These data can be extrapolated to provide reasonable assumptions of decreased levels of expression for most CYP450 enzymes in human body during prolonged space travel. If the prescribed medication regiment is not fully effective or causes undesirable side effects, the ability of the astronauts to function and maintain peak performance levels during space flight could be seriously compromised. Therefore, technologies capable of predicting and managing medication side effects, interactions, and toxicity of drugs during spaceflight are needed. We propose to develop and customize for NASAs applications available on the market Personalized Prescribing System (PPS) that would provide a comprehensive, non-invasive solution for safer, targeted medication management for every crew member resulting in safer and more effective treatment and, consequently, better performance. PPS will function as both decision support and record-keeping tool for flight surgeons and astronauts in applying the recommended medications for situations arising in flight. The information on individual drug sensitivity will translate into personalized risk assessment for adverse drug reactions and treatment failures for each drug from the medication kit as well as predefined outcome of any combination of them. Dosage recommendations will also be made individually. The mobile app will facilitate ease of use by crew and medical professionals during training and flight missions.

Personalized Medication↗
NASA NTRS1998

Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal reorganization. However the underlying mechanism for these cellular changes have not been clearly defined. We examined alterations in endothelial migration, and cytoskeleton architecture (microfilamentous f-actin and vimentin-rich- intermediate filaments) following wounding under simulated microgravity. We also examined the possibility that altered signal transduction pathways, involving protooncogenes, may play a crucial role in microgravity-induced retardation of cell migration and alterations in cytoskeletal organization. We hypothesize that, based on the tensegrity theory, cytoskeletal organization respond to gravitational unloading and through this response, cell behavior, function and gene expression are modified.

Melhado, C. D.↗
NASA NTRS1992

Energy requirements for space flight

Both the United States and the Soviet Union perform human space research. This paper reviews data available on energy metabolism in the microgravity of space flight. The level of energy utilization in space seems to be similar to that on earth, as does energy availability. However, despite adequate intake of energy and protein and in-flight exercise, lean body mass was catabolized, as indicated by negative nitrogen balance. Metabolic studies during simulated microgravity (bed rest) and true microgravity in flight have shown changes in blood glucose, fatty acids and insulin concentrations, suggesting that energy metabolism may be altered during space flight. Future research should focus on the interactions of lean body mass, diet and exercise in space, and their roles in energy metabolism during space flight.

Lane, Helen W.↗
NASA NTRS1992

Research objectives, opportunities, and facilities for microgravity science

Microgravity Science in the U.S.A. involves research in fluids science, combustion science, materials science, biotechnology, and fundamental physics. The purpose is to achieve a thorough understanding of the effects of gravitational body forces on physical phenomena relevant to those disciplines. This includes the study of phenomena which are usually overwhelmed by the presence of gravitational body forces and, therefore, chiefly manifested when gravitational forces are weak. In the pragmatic sense, the research involves gravity level as an experimental parameter. Calendar year 1992 is a landmark year for research opportunities in low earth orbit for Microgravity Science. For the first time ever, three Spacelab flights will fly in a single year: IML-1 was launched on January 22; USML-1 was launched on June 25; and, in September, SL-J will be launched. A separate flight involving two cargo bay carriers, USMP-1, will be launched in October. From the beginning of 1993 up to and including the Space Station era (1997), nine flights involving either Spacelab or USMP carriers will be flown. This will be augmented by a number of middeck payloads and get away specials flying on various flights. All of this activity sets the stage for experimentation on Space Station Freedom. Beginning in 1997, experiments in Microgravity Science will be conducted on the Space Station. Facilities for doing experiments in protein crystal growth, solidification, and biotechnology will all be available. These will be joined by middeck-class payloads and the microgravity glove box for conducting additional experiments. In 1998, a new generation protein crystal growth facility and a facility for conducting combustion research will arrive. A fluids science facility and additional capability for conducting research in solidification, as well as an ability to handle small payloads on a quick response basis, will be added in 1999. The year 2000 will see upgrades in the protein crystal growth and fluids science facilities. From the beginning of 1997 to the fall of 1999 (the 'man-tended capability' era), there will be two or three utilization flights per year. Plans call for operations in Microgravity Science during utilization flights and between utilization flights. Experiments conducted during utilization flights will characteristically require crew interaction, short duration, and less sensitivity to perturbations in the acceleration environment. Operations between utilization flights will involve experiments that can be controlled remotely and/or can be automated. Typically, the experiments will require long times and a pristine environment. Beyond the fall of 1999 (the 'permanently-manned capability' era), some payloads will require crew interaction; others will be automated and will make use of telescience.

Bayuzick, Robert J.↗
NASA NTRS2002

Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

Karr, Laurel J.↗
NASA NTRSDate not supplied

A Brief Review of Chaotropic vs. Kosmotropic Salts on the Degree of Saturation of Membrane Fatty Acids: Implications for Preservation of Environmental Biomarkers

Water activity is well known to have a significant impact on habitability, due to the presence of salts. However, salt composition is also important for habitability, as it can impact microbial activity and biomolecular interactions since certain salts are known to disrupt hydrogen bonding. Chaotropic agents disrupt hydrogen bonding in aqueous solutions leading to increased entropy (chaos) whereas kosmotropic agents are compounds that promote hydrogen bonding (order). The Hofmeister series (Fig. 1) shows the ordering of ions in terms of their ability to stabilize and induce precipitation or aggregation of proteins.

M B Wilhelm↗
NASA NTRS1995

Convective flow effects on protein crystal growth

During the fifth semi-annual period under this grant we have pursued the following activities: (1) Characterization of the purity and further purification of lysozyme solutions, these efforts are summarized in Section 2; (2) Crystal growth morphology and kinetics studies with tetragonal lysozyme, our observation on the dependence of lysozyme growth kinetics on step sources and impurities has been summarized in a manuscript which was accepted for publication in the Journal of Crystal Growth; (3) Numerical modelling of the interaction between bulk transport and interface kinetics, for a detailed summary of this work see the manuscript which was accepted for publication in the Journal of Crystal Growth; and (4) Light scattering studies, this work has been summarized in a manuscript that has been submitted for publication to the Journal of Chemical Physics.

Rosenberger, Franz↗
NASA NTRS1993

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

NASA Discipline Cell Biology↗
NASA NTRS2019

Simulated Microgravity Affects Behavior and Cytokine Expression in the Hippocampus of Adult Mice: Influence of Mitochondrial Reactive Oxygen Species

The effects of microgravity, and social isolation on the CNS are poorly understood. We hypothesize that mitochondrial reactive oxygen species (ROS) play an important role in this process. Since mice are social animals, our lab developed a novel social model of hindlimb unloading (HU), enabling us to determine the effects of both social isolation and simulated microgravity. Responses to 30d of HU were compared in wildtype or transgenic MCAT mice who over-express human catalase in mitochondria. Abundance of 4-Hydroxynonenal, Park7 (a redox-sensitive chaperone and sensor of oxidative stress) and corticosterone were measured by ELISA. Cytokines related to inflammation in the hippocampus and in plasma were analyzed by a protein array. Behavioral data was collected over a 24-hour period.Socially housed HU mice were more active and conducted at least two times more exploratory activities, compared to normally loaded mice. Correlation analysis revealed that specific brain and plasma cytokines correspond with specific behaviors. Simulated microgravity and/or social isolation caused changes in cytokine patterns in the hippocampus and in plasma, with significant interaction effects of HU and genotype in expression levels of five cytokines (out of 35). Interestingly, elevation of these generally pro-inflammatory cytokines by HU in WT mice was mitigated in MCAT mice, suggesting a role for mitochondrial ROS signaling in inflammatory CNS responses to microgravity. Interestingly, socially housed mice had also lower level of 4HNE and higher level of Park7 in the hippocampus compared to singly housed animals. The cytokine responses to social isolation were more extensive in brain vs plasma. Further, there was no overlap in the cytokine repertoire regulated in response to microgravity versus, isolation suggesting divergent mechanisms or downstream signaling. These findings implicate a potentially important role for mitochondrial ROS in CNS responses to the challenges posed both by prolonged missions in space and bedrest on Earth

Guttmann, Linda↗
NASA NTRS2001

An active role of the amyloplasts and nuclei of root statocytes in graviperception

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception. c 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

short duration↗
NASA NTRS2016

GeneLab for High Schools: Data Mining for the Next Generation

Modern biological sciences have become increasingly based on molecular biology and high-throughput molecular techniques, such as genomics, transcriptomics, and proteomics. NASA Scientists and the NASA Space Biology Program have aimed to examine the fundamental building blocks of life (RNA, DNA and protein) in order to understand the response of living organisms to space and aid in fundamental research discoveries on Earth. In an effort to enable NASA funded science to be available to everyone, NASA has collected the data from omics studies and curated them in a data system called GeneLab. Whilst most college-level interns, academics and other scientists have had some interaction with omics data sets and analysis tools, high school students often have not. Therefore, the Space Biology Program is implementing a new Summer Program for high-school students that aims to inspire the next generation of scientists to learn about and get involved in space research using GeneLabs Data System. The program consists of three main components core learning modules, focused on developing students knowledge on the Space Biology Program and Space Biology research, Genelab and the data system, and previous research conducted on model organisms in space; networking and team work, enabling students to interact with guest lecturers from local universities and their fellow peers, and also enabling them to visit local universities and genomics centers around the Bay area; and finally an independent learning project, whereby students will be required to form small groups, analyze a dataset on the Genelab platform, generate a hypothesis and develop a research plan to test their hypothesis. This program will not only help inspire high-school students to become involved in space-based research but will also help them develop key critical thinking and bioinformatics skills required for most college degrees and furthermore, will enable them to establish networks with their peers and connections with university Professors that may help them achieve their educational goals.

genelab↗
NASA NTRS2000

Preparation and Characterization of Fluorescent Derivatives of Chicken Egg White Lysozyme

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow, cascade blue, and 5-(2-aminoethyl)aminonapthalene-l-sulfonic acid (EDANS) have been attached to the side chain carboxyl of asp101 using a carbodiimide coupling procedure. asp101 lies within the active site cleft, and it is believed that the probes are at least partially "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to hisl5, located on the "back side" of the molecule relative to the active site. The fluorescently labeled protein is readily purified from the starting material by cation exchange chromatography. All the derivatives fluoresce in both the solution and the crystalline states. Fluorescence characterization has focused on determining the bound probe quantum yields, lifetimes, absorption and emission spectra, and quenching by added solutes in comparison to the free probe. No appreciable changes are found in the lifetimes of any of the probes except for cascade blue, where Tau(sub free) = 3.52 ns vrs Tau(sub bound) = 2.8 ns. Spectral shifts are found in most cases. Particularly strong quenching upon binding is found in the case of the cascade blue derivative, likely due to probe interactions with the active site cleft. While none of the asp101 bound probes are well quenched by commonly employed solutes, such as potassium and sodium iodide, acrylamide, primuline, the chloride salts of manganese, cesium, and cobalt, trifluoroacetamide, trichloroethanol, and thallium iodide, in those cases where quenching is observed the bound probe is less efficiently quenched relative to the free probe. This indicates that the bound probes are less accessible to the bulk solution, an expected finding for attachment within the active site cleft. Attempts have been made to bind other molecules to these sites, with varying success. Interestingly, all three probes contain one or more sulfonate ((Ar-S03)-) groups. We have not been successful in binding analogous probes without sulfate groups such as pyrene, or with derivatized sulfonate groups such as dansyl type probes, analogous to MANS but where the sulfonate group is derivatized, Ar-S02-N2C2H7. None of the probes is rigidly bound to the protein, i.e., they all have a probe motion superimposed on that of the protein.

Sumida, John↗