Browsing by Subject "Enzymes and Coenzymes"
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Item Advanced Problem Solving in the Biotherapeutics Industry: Parameters influencing the delivery of a novel cell therapy product and exploration of a new method for determining activity of Clostridium histolyticum collagenase, a wound debridement enzyme(2015-05-01) Harris, Melanie A.; Jerry W. Simecka; Patricia A. GwirtzBiotechnology is a multi-faceted industry with many unique challenges that require knowledge in a broad range of topics. When working in the wound care field it is necessary to not only create a product in the laboratory, but also effectively bring it to the patient. This task requires many skilled people who can test it for efficacy, design and conduct clinical trials, confirm quality and consistency, design packaging, consider transportation issues and so on. The following investigation focuses on the testing of a cellular product and its accompanying device under various conditions as well as the exploration of a new assay capable of the activity of a wound debridement enzyme. The results of the product/device testing have generally confirmed the comparability of the cellular product devices as well as their resistance to various temperatures encountered in the clinical environment. A new modified assay for the testing of collagenase has been established as precise and comparable to current methods, though it requires more testing to confirm robustness.Item Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia Pastoris Yeast Cells(2004-05-01) Tchedre, Kissaou T.; Caffrey, James L.; Harris, Ben G.; Wu, Ming-ChiTchedre, Kissaou T., Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia pastoris Yeast Cells (Biomedical Sciences), May, 2004, Lecithin: cholesterol acyltransferase (LCAT) is a key enzyme in mammalian lipoprotein metabolism. Associated with the surface of high-density lipoproteins (HDL), LCAT contributes to the homeostasis of circulating free and esterified cholesterol via the reverse cholesterol transport pathway. The purpose of these studies was to characterize a recombinant form of LCAT, secreted by a human lung cell line (Beta gene 1069/111) and to evaluate a new expression system for LCAT using transformed Pichia pastoris cells. A human lung cell line (Beta gene 1069/111), transfected with pBIISK (Stratagene)+ vector was used as the source of recombinant (rLCAT) for the first stage of characterization studies. Human lung cells were expanded in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum for the expression of the recombinant LCAT. At 80 – 90% confluency, the medium was changed to a serum free preparation and the flasks were incubated for 48 hrs at 37°C to facilitate the secretion of the enzyme. Beta gene (1069/111) LCAT was purified from the conditioned medium using phenyl sepharose chromatography. The purified enzyme was characterized according to: carbohydrate composition, and enzyme kinetic parameters. The enzymatic characteristics, of the human lung cell line LCAT had similar Km and Vmax values to other LCAT preparations, isolated from other expression systems and human plasma. Deglycosylation reduced the molecular weight of the enzyme from about 67,000 to about 43,000 suggesting a carbohydrate component of 25-32% of the enzyme’s total mass. Detailed analysis of the carbohydrate structures revealed N-glycan structures in a complex pattern of sialylated and fucosylated tri and tetra-antennary glycosides (8). In addition to the Beta gene expression, a Pichia pastoris yeast expression system was also developed consisting of human LCAT cDNA cloned into pPICZαA vector along with a removable amino-terminal polyhistidine tag. The Pichia pastoris cells were transformed with a vector containing the LCAT gene cDNA and transformants were selected on agar plates containing zeocine (100μg/ml). Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were used to confirm the correct integration of the LCAT gene cDNA into the pPICZαA vector. The recombinant LCAT produced by the yeast cultures was purified by Talon affinity chromatography, taking advantage of the removable histidine tag. The enzymatic activity was determined using proteoliposome vesicles. The Yeast expression system yielded ~18 mg of enzyme protein/500 ml and thus may provide an appropriate enzyme source for characterization studies via NMR analysis and x-ray crystallography.Item Creating novel purification and biochemical characterization protocols for C. collagenase from Clostridium histolyticum, developing a new emergency medicine product, and formulating several novel therapies for chronic and acute wound treatment(2017-05-01) Mars, Jason P.; Jerry W. Simecka; Patricia A. Gwirtz; Aleksa JovanovicThe pharmaceutical industry not only includes infinite areas of specialization, but also consists of distinct areas that do not typically overlap. Biotechnology is the branch of medicinal research that bridges the gap between the fields within the pharmaceutical industry by being able to take on the challenges that require knowledge of a vast range of information. This practicum was organized to put the scientific knowledge and the interdisciplinary practices of biotechnology to use in a modern day, pharmaceutical company specializing in wound therapy and skincare: Smith & Nephew Biotherapeutics. Wound therapy has the widest range of application due to being one of the few fields that affects everyone, regardless of medical disposition. The specific goals of this practicum were: to develop novel purification and biochemical characterization protocols for C. collagenase from Clostridium histolyticum to replace current production methods of Santyl®, to develop a working prototype of a venom-based, hemostatic film, and perform reformulation, quality control, troubleshooting, and verification testing on samples of Regranex®, Iodosorb “Max”, and EU-Collagenase. Every goal presented was approached with the end results of saving Smith & Nephew costs, reducing bioburden of production, and creating more efficient protocols to bring Smith & Nephew into the modern age.Item Effect of Collagenase Type 2 and Proteinase K Digestion on DNA Yield from Bone Samples Purified on the EZ1 Advanced XL(2015-08-01) Barrett, Lisa C.; Arthur J. Eisenberg; Joseph E. Warren; Raghu R. KrishnamoorthyGenetic results from bone samples often yield low quantities of DNA and poor quality of genetic data. Proteinase K is a proteinase that is commonly used in DNA extraction methods, however the target proteins of Proteinase K do not closely align with the makeup of bone. Collagenase Type 2 is a protease that is more specific to the breakdown of collagen, which bone is comprised of. This study looked at the potential effects of Collagenase Type 2 digestion on bone samples compared to the effects of Proteinase K on quantity and quality of genetic typing. This study also incorporates the EZ1 Advanced XL purification platform and the AmpFLSTR Globalfiler Amplification Kit.Item Elucidation of Mechanism and Molecular Determinants Important in Picrotoxin Action in the 5-Hydroxytryptamine Type 3 Receptor(2003-09-01) Das, Paromita; Basu, Alakananda; Forster, Michael J.; Luedtke, Robert R.Das, Paromita, Elucidation of mechanism and molecular determinants important in picrotoxin action in the 5-hydroxytryptamin type 3 receptor. Doctor of Philosophy (Pharmacology and Neuroscience), September 2003, pp. 192, 3 tables, 26 illustrations, 67 titles. The 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels (LGIC), which mediate fast neurotransmission. Till date, only two subtypes of the receptor i.e. 5-HT3A and 5-HT3B have been investigated. The GABAA receptor antagonist picrotoxin inhibits other anion-selective members of the LGIC. Whether PTX inhibits the cation-selective 5-HT3 receptors was previously unknown. Thus, the primary goal of this study was to elucidate the mechanism of action of PTX and identify the amino acids involved in the action of PTX in 5-HT3 receptors. The overall hypothesis tested was that PTX inhibits the 5-HT3 receptor by interacting in the ion channel. PTX-mediated blockade of the 5-HT3A receptors was non-competitive and use-facilitated similar to GABAA receptors suggesting a conserved site of action of these ligands. The inhibitory effect of PTX was reduced drastically in heteromeric 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in native tissue and recombinant receptor preparations. In anion-selective ion channels, the 2’, 3’ and 6’resides in cytoplasmic aspect of TM2 are known to modulate PTX sensitivity. While mutation of 2’ and 3’ residues in 5-dramatic loss of sensitivity to PTX in 5-HT3A receptors. A converse mutation at 6’ residue in the 5-HT3B subunit caused gain of sensitivity to PTX, suggesting that 6’ is a key determinant of PTX sensitivity. A novel finding was the involvement of 7’ residue in increasing PTX sensitivity in 5-HT3A but not the 5-HT3B subunit. The lack of specific binding by radioligand [3H]EBOB in 5-HT3A receptors suggested that the site of action of convulsants may be different from that anion-selective receptors. The overall results suggest that PTX interacts from that in the anion-selective receptors. The overall results suggest that PTX interact in the ion channel in the 5-HT3 receptors but also underscores the complexity of its interaction with LGICs.Item Exercise-Evoked Metabolic Adaptations in Canine Myocardium(1999-12-01) Stuewe, Steven Richard; Robert Mallet; Neeraj Agarwal; Patricia GwirtzStuewe, Steven Richard, Exercise-Evoked Metabolic Adaptations in Canine Myocardium. Doctor of Philosophy (Biomedical Sciences), November 1999; 128 pp; 4 tables; 17 figures; bibliography, 130 titles. Aerobic exercise training evokes adaptations in the myocardial contractile machinery that enhance cardiac functional capabilities, and the myocardium’s capacity to consume energy. Despite considerable investigative effort, the effects of exercise training on myocardial intermediary metabolism, the source of energy for cardiac function, have not been defined. The investigations described herein were undertaken to delineate the effects of aerobic exercise training on key rate-controlling enzymes of myocardial intermediary metabolism and energy transport, and to characterize the effects of acute exercise on cardiac messenger RNA transcripts encoding metabolic enzymes. To address these questions, dogs were conditioned by a 9 wk treadmill running program or cage rested for 4 wk. Exercise conditioning was documented by a significant decrease in heart rate at rest and during submaximal exercise. A panel of glycolytic and oxidative enzymes was measured in myocardial extracts. It was demonstrated that aerobic exercise training of dogs selectively increased capacities of key rate-controlling enzymes of each of the major pathways of intermediary metabolism in ventricular myocardium. In addition, it appeared that the training-evoked increases in enzyme activities were due to increased enzyme contents, not to changes in substrate affinity. The same training program was implemented to investigate the effects of aerobic exercise training on the myocardium’s energy shuttling system. Total creatine kinase (CK) activity and content of the CKMB isoenzyme were measured in canine myocardial extracts. It was demonstrated that aerobic exercise training increased total myocardial CK activity and CKMB content, although the CKMB isoenzyme remained minor component of the myocardial CK system. A third investigation was conducted to examine the effects of aerobic exercise on the abundance of messenger RNA (mRNA) encoding key enzymes involved in myocardial energy production and transport. Left ventricular myocardium was sampled 30 min after an exercise bout, and messenger RNA transcripts were analyzed by reverse transcriptase polymerase chain reaction. Exercise increased in the myocardial abundance of mRNA transcripts encoding glyceraldeheyde 3-phosphate dehydrogenase, citrate synthase, and the CK-M subunit. These mRNA enhancements could be responsible, at least in part, for exercise-evoked adaptations in myocardial metabolic enzymes demonstrated in the first two investigations.Item Immunoreactivity of Lecithin: Cholesterol Acyltransferase (LCAT); A Tool for Measurement of Levels and Characterization(2000-08-01) Murray, Karen R.; Lacko, Andras G.; Rudick, VictoriaMurray, Karen R., Immunoreactivity of Lecithin:Cholesterol Acyltransferase (LCAT); a Tool for Measurement of Levels and Characterization. Doctor of Philosophy (Biomedical Sciences), August, 2000, 162pp., 12 tables, 30 illustrations, bibliography, 150 titles. Lecithin:cholesterol acyltransferase is secreted by the liver into the plasma where it catalyzes the esterification of high density lipoprotein (HDL) cholesterol as part of the reverse cholesterol transport pathway. Via this pathway both HDL and LCAT have been linked to reducing the risk of atherosclerosis and coronary heart disease. These studies seek to develop an immunoassay to measure LCAT mass and to use immunoreactivity to elucidate the contribution of the highly conserved 121-136 domain of LCAT toward enzyme structure/function and HDL interaction. Several immunoassay models and antibody combinations were investigated to develop an ELISA assay for LCAT. Solid phase immunoassays were found to be most suitable for measuring LCAT from cell culture medium and in partially purified preparations. The immunoassay was analyzed for matrix interference, recovery studies, intra-run precision and inter-run precision. Evaluation of the immunoassay models and antibodies was extended to determine the potential for application toward measuring LCAT in plasma; however, the antibodies screened lacked the needed sensitivity. Studies were conducted to characterize the 121-136 region, a putative lipoprotein substrate binding domain. Differential immunoreactivity was demonstrated for a site directed antibody against the 121-136 region, in contrast to antibodies directed against the entire LCAT molecule, when the enzyme was bound to a hydrophobic surface or to substrate HDL, but not when bound to an alternate antibody. Three naturally occurring mutants within the 121-136 region, were tested for immunoreactivity with the same panel of antibodies and compared to wild type enzyme. These studies demonstrate that the 121-136 region of LCAT resides on the surface of the enzyme that has a high affinity for hydrophobic surfaces and mutation within this region significantly affects the exposure of different epitopes. This suggests that this region could plan an important role in enzyme interaction with its hydrophobic lipoprotein substrates and that mutations within this region could alter enzyme conformation affecting substrate interaction.Item Local Enkephalins Modulate Vagal Control of Heart Rate(2001-05-01) Jackson, Keith E.; James L. Caffrey; H. Fred Downey; Michael W. MartinJackson, Keith E., Local Enkephalins Modulate Vagal Control of Heart Rate. Doctor of Philosophy (Biomedical Sciences), May 2001; 112pp; 7 tables; 22 figures; bibliography, 99 titles. Endogenous opioids, such as enkephalins, were first investigated for their ability to modulate pain. A body of evidence now supports opioid actions in many facets of regulation, including the cardiovascular system. Our laboratory is particularly interested in the ability of opioids to modulate autonomic function. Specifically, the role of the endogenous encephalin, methionine-enkephalin-arginine-phenylalanine (MEAP) was investigated to determine its ability to modulate parasympathetic function in the canine. To investigate MEAP’s response in the sinoatrial (SA) node a novel application of microdialysis was employed, whereby microdialysis was employed, whereby microdialysis probes were fabricated as described by Dr. David Van Wylen (38), and implanted in the SA node. After implantation of the probe, there was a significant attenuation of vagal function during the nodal application of MEAP. Specifically, vagally mediated bradcardia was reduced as compared to control, during the nodal application of MEAP. This inhibition of the vagus by MEAP was blocked by naltrindole, a selective delta antagonist. These data suggested that the vagolytic effects of MEAP were elicited via a delta opioid receptor. To test the hypothesis that MEAP’s effects were elicited through a delta opioid receptor mechanism, selective agonists and antagonists for the opioid receptors were utilized. An attenuation of vagal bradycardia was only observed during the infusion of a very selective delta opioid receptor agonist, deltorphin. A mu and kappa agonist showed no significant differences from control. Deltorphin was observed to elicit vagolytic effects in a similar concentration range as MEAP. However, deltorphin was more efficacious that MEAP. There was a significant attenuation of the deltorphin and MEAP’s vagolytic effects, during the co-infusion of the selective delta antagonist, naltrindole. The mu and kappa antagonists were both ineffective. These data further demonstrate that the observed vagolytic effect is linked to a delta opioid receptor. Endogenous MEAP. A series of experiments were undertaken to determine if endogenous MEAP could be demonstrated in the SA node and is so, was it similarly vagolytic. A preconditioning-like protocol was performed to produce intermittent local nodal ischemia to increase the local concentration of endogenous MEAP. The resulting MEAP was measured and was observed to be elevated during the periods of local nodal ischemia and return to control during reperfusion. Contrary to expectations an augmentation of vagal function was observed, during vagal stimulation. The augmented vagal bradycardia was only observed during ischemia, when MEAP was elevated and returned to control during each subsequent reperfusion. Therefore, there was a correlation between elevated MEAP concentrations and augmented vagal bradycardia. The delta antagonist, naltrindole, prevented the augmented vagal response, during nodal ischemia Glibenclamide, a selective KATP channel blocker, partially reversed the augmented vagal response. These data confirm that delta opiate receptors are involved in the augmented vagal bradycardia and that the mechanism may involve the activation of a KATP channel.Item Mechanistic Studies of O-Acetylserine Sulfhydrylase A from Salmonella typhimurium by Site-Directed Mutagenesis(1996-07-01) Rege, Vaishali D.; Stephen R. GrantRege, Vaishali D., Mechanistic Studies of O-Acetylserine Sulfhydrylase-A from Salmonella typhimurium by Site-Directed Mutagenesis. Doctor of Philospophy (Biochemistry), July 1996, 94 pp., 3 tables, 27 illustrations, bibliography, 64 titles. O-Acetlyserine sulfhydrylase (OASS) is a pyridoxal 5’-phosphate dependent enzyme that catalyzes a β-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide. The pyridoxal 5’-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine. In the present study, the Schiff base lysine was identified as lysine 42 and its role in the OASS reaction was determined by changing it to alanine using site directed mutagenesis. K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates. Apo-K42A-OASS can be reconstituted with PLP suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base. The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the α-aminoacrylate intermediate on addition of OAS suggesting that K42 is involved in the abstraction of the α-proton in the β-elimination reaction. The external aldimine formed upon addition L-ala or L-ser are stable and represent a tautomer that absorbs maximally at 20 nm, while L-cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, also seen in the overall reaction after addition of primary amines to the assay system. The use of a small primary amine such as ethylamine in the assay system. The use of a small primary amine such as ethylamine in the assay system or aminoethylation of C43 in apo-K42A-OASS reconstituted with PLP leads to the initial formation of an internal aldimine followed by the slow formation of the α-aminoacrylate intermediate on addition of OAS. Activity could not be fully recovered suggesting a significant rate enhancement from the presence of K42 for transamination and general base catalysis. Cysteine 43 is the only cysteine in the enzyme OASS-A, and that is next to the internal Schiff base lysine (K42). C43 has been replaced with alanine (C43A), serine (C43S) or threonine (C43T) by site-directed mutagenesis. Also, tryptophan 51 has been replaced with phenylalanine (W51F) or tyrosine (W51Y) by site-directed mutagenesis. Tryptophan 51 is one of the two tryptophan residues in the enzyme, and was thought to be responsible for fluorescence energy transfer to the PLP-internal Schiff base. The growth patterns of the strains carrying mutantions were compared with that of strain DW378 of S. typhimurium carrying plasmid pRSM40 with the cysK gene encoding OASS-A with no mutation. The growth patterns of the strains with mutations of C43A and C43S were very similar to that of pRSM40 whereas the strains with mutations at C43A and C43S were very similar to that of pRSM40 whereas the strains carrying mutations at W51F, W51Y and C43T showed very slow growth. The purification procedures for all of the mutant enzymes were similar to that of the wild type enzyme.Item Protein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B(2003-12-01) Confer, Nils Forgard; Alvarez, Rafael; Ben S. Aar…; Wu, MingehiConfer, Nils Forgard, Protein-Protein Interactions Between Poly(ADP-ribose) Polymerase-1 and DNA Polymerase B. Doctor of Philosophy (Biomedical Sciences), December 2003, 114 Pages, 22 Figures, 1 Graph, and 80 References. The mammalian genome is continually subjected to chemical and environmental modifications that are repaired by base excision, and when excessive, may lead to apoptosis. Interestingly, the chromosomal enzyme poly(ADP-ribose) polymerase-1 (PARP-1) appears to modulate both mechanisms, either facilitating DNA repair and/or modulating cell death. In this dissertation project, experiments were performed to address the regulatory potential of PARP-1 in base excision repair (BER) and specifically on DNA polymerase B (pol B) function. Activity gels were used to measure the DNA polymerase activity of pol B following protein-(ADP-ribosyl)ation. However, the fraction of pol B molecules (ADP-ribosyl)ated was never 100% under the reaction conditions employed. In fact, similar results were observed in activity gels specific for PARP-1, even under conditions where this polymerase is the primary nuclear acceptor for poly(ADP-ribose) Here, I also describe a newly developed electrophoretic-mobility-shift-assay (EMSA) to monitor for the specific binding of pol B to a custom-made five-nucleotide gapped DNA duplex. However, while specific for pol B, this assay was inefficient to monitor the effects of covalent poly(ADP-ribosyl)ation on pol B activity. Moreover, I also observed the specific molecular association of PARP-1 is specifically proteolyzed into peptide fragments by caspases, conditions were established for the efficient proteolysis of PARP-1 by either capase-7. Experimental results indicated that caspase-3 was more efficient than caspase-7 at splitting unmodified PARP-1 into two peptide fragments. By contrast, caspase-7 appeared best suited for the proteolysis of covalently auto-poly(ADP-ribosyl)ated-(PARP-1). Interestingly, both of the caspase-generated peptide fragments of PARP-1 specifically associated with pol B as supported by co-immunoprecipitation/immune-blotting experiments. Taken together, the experimental results presented here support the hypothesis that a molecular mechanism exists that involves interaction(s) of PARP-1 with pol B that may help to facilitate the decision making process between cell survival and cell death. Thus, upon proteolytic degredation of PARP-1 into a 24-kDa amino-terminal fragment and an 89-kDa carboxy-terminus, each truncated peptide, separately, retains physical association with pol B, and inhibits DNA repair associated pol B activity to irreversibly switch the fate of cell from BER toward chromatin degradation and, eventually, programmed cell death.Item Sensing and imaging of hyaluronidase activity using a long-lived fluorophore(2016-05-01) Chib, Rahul; Gryczynski, Zygmunt; Fudala, Rafal; Borejdo, JulianThis dissertation explores the synthesis, characterization and biomedical applications of a fluorescent probe for sensing and imaging of hyaluronidase activity. The enzyme hyaluronidase is overexpressed in various cancer including bladder cancer, prostate cancer, melanoma, head and neck carcinoma etc. Fluorescence-based sensing and imaging have tremendous applications in biomedical sciences. A fluorescent probe specific to a disease biomarker can help in the diagnosis and treatment of various diseases like cancer. Fluorescence emission in the red region of the electromagnetic spectrum provides the best optical window for sensing and imaging, as the contribution of autofluorescence decreases in this region. To distinguish the signal from the fluorophore and autofluorescence, efforts have been focused on developing red-emitting fluorophores, preferentially with a long fluorescence lifetime (significantly longer than autofluorescence). This improves signal-to-noise ratio and opens the possibility for time-gated detection. However, the commercially available red fluorophores have a very short fluorescence lifetime. The groups of currently developed triangulenium fluorophores like Azadioxatriangulenium (ADOTA), which emits in the orange/red region with a long fluorescence lifetime and high quantum yield presents great opportunities for sensing and imaging applications. The goal of this study was to characterize the photophysical properties of Azadioxatriangulenium (ADOTA) fluorophore and explore its properties for biomedical sensing and imaging. A sensor for the enzyme hyaluronidase was developed by using ADOTA fluorophore. This sensor was developed by heavy labeling of hyaluronic acid with ADOTA fluorophore. Results from these studies show the applications of ADOTA in fluorescence-based sensing, as a contrast agent in fluorescence-lifetime imaging microscopy (FLIM), and its application in time-gated detection for background-free cellular imaging.Item The 6' and 7' Residue of the Second Transmembrane Domain of Ligand-Gated Ion Channels Influence Gating and Picrotoxin Sensitivity(2005-07-01) Gonzales, Eric B.; Dillon, Glenn; Luedtke, Robert R.; Martin, MichaelGonzales, Eric B., The 6’ and 7’ residue of the second transmembrane domain of ligand-gated ion channels influence gating and picrotoxin sensitivity. Doctor of Philosophy (Pharmacology and Neuroscience), July 2005, pp213, 4 tables, 33 illustrations, 89 titles. The GABAA and glycine receptor are members of the Cys-loop family of ion channels. These receptors mediate rapid neurotransmission in the nervous system. Picrotoxin (PTX) interacts within the channel near the TM2 2’-6’ position, with the most critical interaction at the 6’ position. The present studied addressed the stoichiometric dependence and molecular requirements of the TM2 6’ position on PTX sensitivity. I hypothesized that there is not a stoichiometric dependence and that residues with a hydroxyl group remain sensitive to PTX. Further, work previously completed in the laboratory demonstrated that the TM2 7’ position influences channel kinetics in the serotonin type-3 receptor. However, similar work has not been performed in other members of the Cys-loop family of receptors. I hypothesize that the TM2 7’ position influences both gating in the glycine α1 receptor similarly to that in the serotonin type-3 receptor. Additionally, the TM2 7’ position could influence the PTX in the glycine α1 receptor. Picrotoxin sensitivity was determined to not be stoichiometrically dependent on the subunit location of the T6’F mutation in α1β2 or α1β2γ2 GABAA receptors, a single T6’F mutation was sufficient to eliminate PTX sensitivity. The α1(T6’F) β2 receptor showed PTX concentration-dependent stimulation. Picrotoxin sensitivity had a rank order of potency in α1β2(mutant) as follows: Serine [greater than] Threonine = Alanine [greater than] Cysteine [greater than] Tryptophan. In several cases, the kinetics of the T6’F mutant receptors exhibited rapid desensitization during prolonged application of agonist. In combinations of subunits with the T6’F mutation, two or fewer mutant receptors appeared to have normal phenotypes. Three or four T6’F mutations exhibited rapid desensitization, and strongly suggests that the stoichiometery of the α1β2 GABAA receptor is two α subunits and three β subunits in the receptor. The Gly α1(T7’L) mutation exhibited enhanced glycine sensitivity with slower gating kinetics than the wild type (approximately 3-fold slower); the T7’A mutation had significantly reduced glycine affinity. The glycine EC50 kinetics of the α1(T7’A) mutant receptor was more complex than the wild type. There appears to be a complex interaction between agonist binding and gating of the channel that is disrupted by the 7’ position mutations. Picrotoxin sensitivity in the mutation were enhanced with either a T7’A or T7’L substitution. The data suggest that the 7’ residue may play an accessory role in shaping the PTX site. Finally, these residues are also critical in gating of the receptor. Residues critical for gating and PTX sensitivity may be coupled in the normal function of the Cys-loop family of receptors.